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Gardens’ Bulletin
Singapore

EB 19 7008

VOL.59 (Parts 1 & 2) 2007 ISSN 0374-7859

Proceedings of the 4th International Ginger Symposium

34 - 6" July 2006
(Editors B.C. Tan and J. Leong-Skornickova)

Singapore Botanic Gardens

THE GARDENS’ BULLETIN SINGAPORE

The Gardens’ Bulletin Singapore publishes original papers on plant taxonomy
(including revisions), horticulture, phytogeography, floristics, morphology,
anatomy and related fields with emphasis on plants in the West Malesian
region.

. Dr. B. C. Tan Dr. Jana Leong-Skornickova
Singapore Botanic Gardens Singapore Botanic Gardens
(Editor) (Assistant Editor)
Dr PX. fan Ms. C. Soh
National Parks Board Singapore Botanic Gardens
(Assistant Editor) (Journal Business Manager)
EDITORIAL BOARD
Dr-S:C. Chin Dr. M.C. Roos

Singapore Botanic Gardens
Singapore

Dr. M.J.E. Coode
Royal Botanic Gardens
Kew, U.K.

Prof. Sir P. Crane
University of Chicago
Chicago, USA

Dr. R.T. Corlett
University of Hong Kong
Hong Kong

Dr. W. J. Kress

Department of Botany, NMVNH

Smithsonian Institute
Washington DC, USA

National Herbarium Netherlands
Leiden University, The Netherlands

Dr. E. Soepadmo
Forest Research Institute Malaysia
Kepong, Malaysia

Prof. T. Stuessy
University of Vienna
Austria

Dr. W.K. Tan
National Parks Board
Singapore

Dr. I. M. Turner
Research Associate
Singapore Botanic Gardens

The Gardens’ Bulletin is published twice yearly by the National Parks Board, Singapore.
Neither the National Parks Board nor the Editorial Board is responsible for the opinions or
conclusions expressed by the contributing authors.

The annual subscription for the Gardens’ Bulletin is Singapore $100.00 including postage.
Overseas subscribers are required to make payment in the form of bank drafts or international
money orders in Singapore currency payable to National Parks Board, Singapore.

Instructions for contributing authors are found on the inside back cover.

[Cover photo : Etlingera coccinea (see p.183); photo by A.D. Poulsen]

3 FEB 19 tht ie

Larary

The Gardens' Bulletin

Singapore

VOL. 59 (Parts 1&2) 2007 ISSN 0374-7859
CONTENTS
K. Larsen
The exploration of gingers in SE Asia - some milestones and perspectives ............:ccccsceeseeees 1
A.M. Aguinaldo
Selected Zingiberaceae species exhibiting inhibitory activity against Mycobacterium
PDETCHIOSTS ie Any amp ily TOCME MICA HOMIE 52. .escccos- wsesqastennnsenendssasedennandvecucasncsstnesnsaceeabnrbacce 13

B.B. Bau and A.D. Poulsen
Ethnobotanical notes on gingers of the Huon Peninsula in Papua New Guinea ............. oo

H.H. Bay, H.B. Sani, P.C. Boyce and S.L. Sim

Rapid Jn Vitro Propagation of Hornstedtia reticulata (K. Schum.) K. Schum. ..............0. a5
L.B. Cardenas

Variations in tissue development and secondary product elaboration of Hedychium

coronarium J. Konig floral cultures grown on different Media ........ cece eeeeeeseeeeeneeeeeeeee 41

E.W.C. Chan, Y.Y. Lim and T.Y. Lim
Total phenolic content and antioxidant activity of leaves and rhizomes of some ginger
Se cles wey Pe mimcmlle ya DNS seer e seat cee tear sare teces cocky sactiond¥ecnenedsdeaisneds vosadsnnuscveseessosnuevondeaseye 47

M. Dan, B. Sabulal, V. George and P. Pushpangadan
Studies on the rhizome oils from four Hedychium species of South India: a
CS HN Tet OM co UU ese TCO AC Mr eee ee ccs cnet ae eo raat recs cuizes nsvesisecadodeeevpsmncssniaxonsosucduula don peandeneee 57

M.N. Hamirah, H.B. Sani, P.C. Boyce and S.L. Sim
Micropropagation of Boesenbergia pulchella Ridl., a potential ornamental plant ......... 65

H. Ibrahim, N. Khalid and K. Hussin
Cultivated gingers of Peninsular Malaysia: utilization, profiles and micropropagation ......

R. Johns
An introduction to the New Guinea database, with notes on the Zingiberaceae, specifically
eee lca (O) inemmre eae rete etree ete tere te PAE ase eect aun dssads cr ipa Saeeatawnnionane sudden eon Sue atesOesestes 89

W. Kaewsri and Y. Paisooksantivatana
Morphology and palynology of Amomum Roxb. in Thailand 0.0... eeeseeseesseeseeeseeees 105

W.J. Kress, M.F. Newman, A.D. Poulsen and C. Specht
An analysis of generic circumscriptions in tribe Alpinieae (Alpinioideae: Zingiberaceae)
scsusensi soscubensubldasdivebieduensaesbecueetnchiastouscaecadsi dial eenetlits ott esas enaMeee ae ne i ice a 113

K.H. Lau, C.K. Lim and K. Mat-Salleh
Materials for a taxonomic revision of Geostachys (Baker) Ridl. (Zingiberaceae) in

Penmsular Malaysia ...........:s<sss-ssssssscvscssssaeseneshanevederesteomautatoneesstner tem eeNne teem ear 129
M.F. Newman

Materials towards a revision of Aulotandra Gagnep. (Zingiberaceae) ...........:ccccceeeeee 139
A.D. Poulsen

Eilingera Giseke Of Java ssscsgeccectietencsisensactansssiuctehagecdy nce ee saeeee eee ee eae ee 145

S. Ruamrungsri, J. Uthai-butra, O. Wichailux and P. Apavatjrut
Planting date and night break treatment affected off-season flowering in Curcuma
alismmatifolia Gagme ps xiis.csaaeieeepsestbectes siasnascoseesnecceennee tacts eee nee eee ae en 173

P. Saensouk, P. Theerakulpisut, B. Kijwijan and S. Bunnag
Effects of 2,4-D on callus induction from leaf explants of Cornukaempferia larsenii
Saensouk, 110771. SCHOG io icic ca sdekec ke veccoasuscnsntsssuceesee sees sae ee 183

G.J. Sharma, P. Chirangini and K.P. Mishra
Evaluation of anti-oxidant and cytotoxic properties of tropical ginger, Zingiber
montanume: (J.KOmIg) AG DICE, oo .csccsncsncscienensoceetecereeettee cent te tae eeee eee ee 189
P. Sirirugsa, K. Larsen and C. Maknoi
The genus Curcuma L. (Zingiberaceae): distribution and classification with reference to
species diversity in Thala’ is..seszesnesnsice<teconeneeste Meee tee eae ee ne ee 203

J.-J. Song, P. Zou, J.-P. Liao, Y.-J. Tang and Z.-Y. Chen
Floral ontogeny in Alpinia oxyphylla Mig. (Zingiberaceae) and its systematic significance
nsanedahdsnnanonianesdapiinceogaesnteundearassdnungpbede sedan dvndiegtTteian ethos gee mete eee eee eee 221

Date of publication: December 2007

Published and copyrighted by

National Parks Board
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http://archive.org/details/gardensbulletins459unse —

Gardens’ Bulletin Singapore 59 (1&2): 1-12. 2007 i

The Exploration of Gingers in SE Asia — Some Milestones
and Perspectives

Symposium keynote lecture

K. LARSEN

Department of Systematic Botany, University of Aarhus, Building 1540 DK-8000
Aarhus C, Denmark

The international symposia on Zingiberaceae are fora for the communication
of new research and meeting places for all who are interested in
Zingiberaceae and related families to exchange ideas and establish new
contacts. Sometimes, it is also worthwhile to look back to former times
and re-evaluate earlier researchers’ works and to consider what we have
achieved and what should be done. How can we, in the light of what has
been done, do better in the future? How can we make the most of what are
the available resources - human and economic. That is what I will try to do
here today, hoping that it may be useful in our exchange of ideas during this
meeting here in Singapore, which is and has been, for more than a century,
one of the centres of research in taxonomy of Zingiberaceae in SE Asia, the
region I know best and on which I will concentrate my speech.

One of the first scientific collections of gingers was made by the
German medical doctor Engelbert Kaempfer who visited Thailand in 1690
on his way to Japan. We do not know much about his collections but Linnaeus
described two small plants after him, Kaempferia galanga and Kaempferia
rotunda. Linnaeus enumerated rather few species of Zingiberaceae in his
Species Plantarum (1753), the starting point for botanical nomenclature.
Besides these two species of Kaempferia we find Zingiber zerumbet which
he referred to the genus Amomum, Elettaria cardamomum also mentioned
as an Amomum species, and two Curcuma species, Curcuma longa and
Curcuma rotunda, the latter now referred to Boesenbergia.

Johan Gerhard Koenig was another early explorer in SE Asia. He was
German-Baltic by birth, studied for a few years under Linnaeus and became
a Danish citizen before being sent as a medical doctor to the Danish colony
Trankebar in India in 1767 where he undertook large collections. In 1779 he
went to Siam and in 1781 to Ceylon. He became a good friend of Roxburgh
who also looked after him during his last illness. He was one of the most
important collectors in the 18 century even though he published little himself.
He described 21 Zingiberaceae based on living material. The descriptions
were sent to his friend Retzius for publishing in 1783. K. Schumann

Gard. Bull. Singapore 59 (1&2) 2007

characterized Koenig’s descriptions as excellent. Koenig established 4 new
genera, Hura (Globba), Languas (Alpinia) , Hedychium (H. coronarium)
and Banksea, later changed to Costus and Costus malaccensis Koenig. His
Siamese collections were mainly from the island Yunk Ceylon, not to be
mistaken for Sri Lanka, it is what today is known as Phuket in Thailand.
His large collection of manuscripts was, thanks to Roxbourgh, given to Sir
Joseph Banks, and is now bound in 21 large volumes in the British Museum;
herbarium specimens are found there and in Copenhagen. In literature at
least up to 1980, and maybe later, we read that Koenig’s collection from
Siam was lost. They were, however, rediscovered in Copenhagen Botanical
Museum some 20 years ago.

Real exploration of the family Zingiberaceae began in the 19"
century with important works, such as Roscoe’s Monandrian plants from
1828. Roscoe was an English historian, and a botanist in Liverpool. He was
born in 1753 and became partner in a bank where he lost his money and
became bankrupt in 1820. He was also the founder of the botanic garden
in Liverpool. In 1824-28 he published his magnificent work: Monandrian
plants of the order Scitamineae. It was issued in 150 copies, including 112
hand-coloured lithographs. He treated 66 species of Scitamineae most of
which were cultivated in the Botanic Garden.

Important descriptions were undertaken also by Roxburgh at the
Calcutta Botanic Garden and by his successor, the Danish born Nathanial
Wallich (born Nathan Wulff in Copenhagen) who in his magnificent work,
Plantae Asiaticae Rariores, published excellent illustrations of plants from
British India including British Burma, among these several Zingiberaceae,
e.g., Kaempferia candida and Kaempferia elegans, both described from
Burma.

In the Dutch colonies, today’s Indonesia, several botanists were
active, among them C. L. Blume, a German born botanist, who worked for
many years and collected several thousand numbers in Java at the beginning
of the 19 century and later became director of the Rijksherbarium in Leiden.
The Dutch botanish, Friederich Miquel, also made important contributions
and described numerous species from SE Asia. He was a medical doctor
from Groningen, and later director of the botanical garden in Rotterdam
and professor in Amsterdam, finally succeeding Blume as director of Leiden
Rijksherbarium from 1862. From the beginning of the 20 century, Valeton,
another Dutch botanist educated at the university of Groningen, became
one of the most important explorers of the Zingiberaceae mainly from Java.
He first worked in Java at the Dutch sugar cane experimental station but
finally became director of the herbarium in Buitenzorg, now Bogor. He
took a great interest in Zingiberaceae and described numerous species with
very careful and detailed drawings and descriptions from all over the region

The Exploration of Gingers in SE Asia 5

as far.eastward as New Guinea. His drawings are among the finest and most
detailed in the family at that time and also his descriptions are extremely
careful and precise. In his later years he worked at the Rijksherbarium in
Leiden.

Another fine work from the middle of the 19° century is that of
the Russian botanist, Paul EF Horaninov: Prodromus monographiae
Scitaminearum from 1862. This work, although mainly a compilation of
known literature, is still important for its time. He described species not only
from Java, but also from New Guinea and other parts of the Dutch colonies
in SE Asia.

The Italian botanist, Odoardo Beccari, is indeed admirable. He was
born in Firenze in 1843 and died there in 1920. For more than 10 years
he collected in Borneo, the Moluccas, Sumatra and Java, amassing several
thousands of collections. He is perhaps best known for his extensive palm
collections, though his zingiberaceous collections are as important. They are
among the finest and most carefully prepared specimens found in herbaria.
When one takes into consideration the extremely difficult conditions under
which he worked in the field for months they are indeed excellent. They are
today found as a special collection in the herbarium in Firenze where he
became Director in 1876.

If we now try to summarize our knowledge towards the end of the
19" century and look at the comprehensive work of the De Candolles’
Prodromus systematis naturalis regni vegetabilis we find the treatment of the
Zingiberaceae from 1883. Here are enumerated 21 genera with in all ca 250
species. The largest genera are Amomum with 50, and A/pinia with 40 species,
though the circumscription of Amomum does not correspond to ours.

Six years later, in 1889, we get an overview of the Zingiberaceae in
a form that we can compare with today’s systematics. That was done by a
Danish systematist O. G. Petersen, a professor in Botany at the Royal Danish
School of Forestry and Agriculture in Copenhagen, in the first edition of
the ’Die natiirliche Pflanzenfamilien” edited by A. Engler and K. Prantl.
Here, Petersen, who mainly worked with Neotropic Zingiberaceae, treated
24 genera with a total of ca 240 species including what we today recognize
as Costaceae. Just to give an idea of how poorly the SE Asian tropics were
known, the genus Alpinia was estimated to contain about 40 species, while
we today recognize ca 250 species, whilst the other similarly large genus
Amomum had 50 species including what we today treat as Etlingera. Finally
the genus Zingiber was estimated to comprise ca 20 species, about one third
of what we find today in Thailand alone.

The great breakthrough came with three botanists working about
the turn of the century or around 1900, namely Schumann, Ridley, and
Gagnepain.

4 Gard. Bull. Singapore 59 (1&2) 2007

I shall begin with Karl Moritz Schumann. The standard work
on the family for about a century has been the large monograph on the
Zingiberaceae from 1904 by Schumann in A. Engler’s monumental world
flora: ’Pflanzenreich”, in which he recognizes over 800 species. About half
of these were described by Schumann himself from the German colonies
in the eastern part of the area, New Guinea, and the Bismark Archipelago,
but also from Borneo, Sulawesi and other areas. It is remarkable that in
the 15 years from 1889 to 1904, the number of species almost tripled. In
the subsequent 100 years we have been able to describe almost twice as
many species. Holttum, in his treatment of the Zingiberaceae of the Malay
Peninsula or rather Peninsular Malaysia, expressed a very critical attitude
towards Schumann’s work. This is impossible for me to understand and can
only be caused by his anti-German feeling. Schumann’s work is admirable
even if we find today that many of the genera he treated have another
circumscription than the one we now recognize. Let me add that in many
works also in taxonomic literature we can read that Schumann’s types were
lost during the destruction of the Berlin Herbarium in World War II. That is
true only for the herbarium material. The large collections of spirit material
were miraculously saved and many types of Zingiberaceae are still preserved
in the Berlin Herbarium in perfect condition.

In the French colonies several collectors worked all over Indochina in
today’s Cambodia, Laos and Vietnam. The same year as Schumann published
his work, Francois Gagnepain wrote his account for ”’Flore Générale de
l’Indo-Chine”. He based his descriptions on very careful dissections and
fine line drawings. In the herbarium in Paris we still find small envelopes
with the material, the remains of his dissections, very often nicely arranged
and glued to a piece of paper with fine pencil sketches. Gagnepain described
Zingiberaceae not only from Indochina, but from all over tropical Asia.

In 1899, Henry Nicolas Ridley, Director of the Botanic Garden
Singapore, published his account of the Scitamineae of the Malay Peninsula
based on his own and several others’ collections. He described more than
100 species, several of them from the botanical gardens in Singapore and
Penang. But his contribution to the Scitamineae did not end with his work
on the Ginger flora of the Malay Peninsula. He also contributed to our
knowledge of the Zingibereaceae of Borneo in 1906 and the Philippines
in 1909. He also described species from Indochina and Africa. All in all he
described over 300 species. His work was critically discussed in the excellent
paper by I. M. Turner, published in the year 2000, who writes, ” His hurry to
describe the myriad of undescribed taxa he encountered frequently led to
scrappy, inaccurate, or even erroneous descriptions and nomenclatural and
other taxonomic muddles. The mistakes Ridley made during his publishing
career could probably provide all the examples needed for the International

The Exploration of Gingers in SE Asia 5

Code. However, his achievements far outweigh his misdemeanours”, a
sentence that might also be applied to the two other great names, Schumann
and Gagnepain. In this connection we should also remember the words of
Airy Shaw, who in an article unveiled a mistake made by the founder of
Flora Malesiana, the late Prof. Van Steenis, who described a new genus that
turned out to be some monocot leaves mounted with a legume flower. Airy
Shaw’s conclusion was, He who publishes nothing makes no mistakes”.

The next great synthesis came with Loesener’s contribution in 1930
to the second edition of ’ Natiirliche Pflanzenfamilien”. It is, naturally, based
on Schumann’s monograph with the same illustrations, but with a more
”*modern” approach to the generic concept.

Around the middle of the 20 century little was published except for
Holttum’s revision of the Zingiberaceae of the Malay Peninsula published
in Gardens Bulletin Singapore in 1950. It was based on material available
there and mainly collected by Corner in 1930-40. Holttum based his work
on careful dissections of living material as well as on Ridley’s collections.
He paid much attention to inflorescence structures. His descriptions vary
much in length and the drawings are often of little use as they are of a
rather poor quality. His work is, however, still very useful when it comes to
determination of species from the Malay Peninsula even if it also reflects
the fact that Holttum, during the war, had very little access to literature that
was not available in Singapore, something he was himself very well aware of
and that he also told me during many fruitful discussions in the sixties when
I worked in Kew and profited much from his knowledge. He expressed it
once in the middle of the sixties in words along the lines of: ”I wrote down
what I knew about the Zingiberaceae of Malaya at that time”.

Between 1970-90, B.L. Burtt and Rosemary Smith from Edinburgh
did a remarkable amount of work towards a better understanding of the
Zingiberaceae, not least of Borneo. In an early paper they dealt with the
taxonomic history of the classification of the Zingiberaceae, in which they
pointed out the many nomenclatural problems that exist and suggested
solutions. Rosemary Smith contributed several revisions and a fine overview
and a new classification of the largest genus, Alpinia, a work that is still
respected in spite of later molecular work.

The latest overview of the family was published in 1998 in the
”’Families and Genera of Vascular Plants” edited by Kubitzki, following the
tradition of Engler in the new century. Here I treated the Zingiberaceae
in collaboration with J.M.Lock, M. and P. Maas, on the basis of what we
knew at that time. It was just a couple of years before the turn of the
century and the molecular age was just beginning. Two years later Kress
and his collaborators published their new approach to the system of the
Zingiberales and our knowledge took a turn towards a true phylogenetic

6 Gard. Bull. Singapore 59 (1&2) 2007

system. That is where we are today and what we shall hear much more about
at this symposium.

Now we can ask the questions, what have we achieved? How deep
or complete is our knowledge of the Zingiberaceous flora of SE Asia? Let
us take a quick look at the regions.

I have concentrated in this lecture on SE Asia, but I shall just
mention with a few words the situation on the Indian subcontinent where
Indian botanists are now contributing greatly. I am also sure that even if
India belongs to a part of tropical Asia which has been rather well studied
botanically for three centuries, there are still many areas in which undescribed
taxa will be found as we have recently seen, e.g., from the Nicobar Islands,
and the vast mountainous regions in the extreme north and northeast.

If we move from India towards the East the next country we meet is
Myanmar. This was included in Hooker’s ’ Flora of British India” but was far
less collected than India. Today it is hardly possible to do serious collecting
work in Myanmar due to the political situation, as the most interesting areas
cannot be reached. I am, however, not in doubt that when the time comes
that we can freely move around in Myanmar, many new taxa will be found.
During my work with the genus Caulokaempferia | have seen material
collected by George Forrest from the frontier between Myanmar and China
representing more that one undescribed species, but the collections are too
poor to be described properly. Undoubtedly, some of the old taxonomists
would have described these as new species. Still new species have been
described as, e.g., Smithatris myanmarensis W.J. Kress and Mantisia wardii
Burtt & Smith. All in all the number of species documented from Myanmar
is ca 150 and that is according to my experience very low. There is, in my
opinion, no doubt that twice as many species occur in that country.

Let us turn to China where we have a new revision. China is a country
where taxonomy has a high priority as a basic science strongly supported
by the Academy of Science and the government. Chinese botanists are in
these years doing an enormous amount of work collecting, describing and
publishing. The great partnership between the Missouri Botanical Garden
and the Chinese Academy of Science, which is producing the second
edition of the Chinese Flora, now in English, is admirable. I co-authored
the Zingiberaceae myself with 216 species in 20 genera. 141 species or over
the half are endemic. This contribution was published in year 2000. In the
same year my co-author, Dr. Wu Te-lin published one new species of Alpinia
from the Guangdong province and more have been found since. We have
also found recently that some of the supposedly endemic species also occur
in Thailand and others will probably be found in northern Vietnam. So
collecting gingers in the hilly southern tropical provinces of China is far from
complete even if we may regard China as, probably the best studied country

The Exploration of Gingers in SE Asia if

from a Ginger specialist point of view. Also in year 2000 a new species of
the hitherto monotypic genus Vanoverbergia was found and described by
Funakoshi and Ohashi from Taiwan.

The Indochinese countries, Cambodia, Laos and Vietnam, were
treated together by Gagnepain in the ’Flore générale de I’Indochine” in
1908. Comprehensive collecting of Zingiberaceae has not been undertaken
since the publication of that Flora in which 12 genera with, in all, 102
species were recognized; 62 of which were described by Gagnepain. From
the numerous unnamed collections in Paris on which I worked with in the
60s and 70s, I described several new species but it was also clear that much
old material was inadequate for describing. For several decades it has not
been possible to travel to and in these countries. Fortunately the situation
has completely changed and a new generation of botanists is now working
seriously in exploring these countries with international cooperation, e.g.,
between Russian and American taxonomists and the National Museum in
Hanoi.

From this fruitful partnership between the herbarium in St. Petersburg
and Hanoi, the Orchids have been treated. I have seen photographs of
numerous unknown Zingiberaceae collected in the north of Vietnam where
the limestone region seems to be particularly rich in species. From the central
limestone region of the country, Mark Newman several years ago described
the new genus Distichochlamys. A few years ago a Russian zoologist found
a second species, and immediately after, a third species was found. From
Laos a beautiful little Curcuma-related plant was found by Dr. Jenjittikul
at the Chatuchak flower market in Bangkok where loads of plants are
brought from across the border at the Mekong River. We described this as
Laosanthus graminifolius .The plant is now found in nurseries in the USA.
Today we have documented over 200 species from these three countries,
the question is then, is the flora of these three Indochinese countries less
rich than the Thai flora with 300 species?” I do not think that, and I believe
that another 100 species will be added when the rich plant communities,
particularly of Vietnam and Laos, are properly explored.

Peninsular Malaysia and Singapore are, as China, well studied
botanically, but still new species are found. A beautiful species of Haniffia
was recently refound and it was suddenly possible to solve a question about
the occurrence of the genus in Thailand, first posed by Holttum. The plant
mentioned by Holttum is not a Haniffia, but at the same time I collected
a second species from Thailand. A few years ago we published a popular
booklet, which gives an overview of the ginger flora. The time has come,
however, where a full treatment of the Zingiberaceous flora of Peninsular
Malaysia should be published. Iam here thinking of a book similar to that of
Gunnar Seidenfaden & Jeffrey Wood: The Orchids of Peninsular Malaysia

8 Gard. Bull. Singapore 59 (1&2) 2007

and Singapore. With the tradition of ginger research in the Singapore
Botanic Gardens going back to Ridley and Holttum, it would be a fine way
to commemorate these two pioneers.

From these more or less well documented countries we shall move
to a region which is far more difficult to overlook. Coming from Peninsular
Malaysia it might be natural to continue to Eastern Malaysia: Sarawak and
Sabah. Here the situation is different. There are still vast areas to explore as
seen from the many new species described of, e.g., Boesenbergia, Zingiber,
Etlingera, Alpinia, and the new genus, Tamijia. This last genus was found by
molecular studies to constitute its own subfamily Tamijioideae. There is still
a vast field for exploration in this part of Malaysia and I have no doubt that
many new taxa will be found on each new collection expedition there.

Indonesia is even more difficult to deal with, even though we now
have an excellent overview in the checklist published by Newman, Lhuillier
and Poulsen from 2004 covering the whole Malesian area. This vast country
is centred around the island of Java with one of the best documented floras
of the region, not much new could be expected from the national parks here.
Quite different is the situation in Sumatra, which is one of the islands that
should attract more attention. Several new Globbas have been described
and I am aware of new Boesenbergia species which cannot yet be described
due to the poor state of the existing material. This is indeed a problem with
ginger collections made by general collectors who do not know how to
preserve or describe the delicate structures of these plants. East of Java
the challenge becomes even bigger. Few genera have been revised as, e.g.,
Burbidgea, endemic to Borneo. When I say Borneo it is strange that it seems
that there are far more gingers in the northern and eastern Sarawak and
Sabah than in Kalimantan. I am not in doubt that this is more due to lack of
collecting than to phytogeographic peculiarities. The more we go East in the
Malesian region the poorer is our knowledge. New Guinea and the Bismark
Archipelago have not been visited for decades by systematic collecting
expeditions concentrating on Zingiberaceae. We are here practically at the
same level of information as 100 years ago when Schumann published his
work. His short descriptions are often difficult to evaluate. Just to give
an idea of the situation let me mention the genus Riedelia with about 100
species, which has never been revised and with numerous species only known
from the type locality. In the genus Plagiostachys, much of the old materials
are useless as the flower structures in many species cannot be seen due to
the deterioration of the inflorescence in a slimy substance after flowering.
Even though a survey of the Bornean species was published by Rosemary
Smith in 1985, we know that it will be a long time before a revision can be
written. What I have said about these two genera could be repeated in the
case of Pleuranthodium.

The Exploration of Gingers in SE Asia 9

The Zingiberaceae of the Philippines were treated by Ridley in
1909 based on specimens in the herbarium in Manila and the numerous
collections made by Elmer and Merrill and also the classic collections by
Haenke, Cuming and Blanco. Again here many species are only known from
the type locality or very few collections.

Let me end this very short presentation of the SE Asian regions
with a more comprehensive review of the status of Thai Zingiberaceae, the
area I know best, and a flora with which I have worked for half a century. I
still remember my first collection of the family from SE Thailand in January
1958. My field notes read, ’small, terrestrial orchid with green flowers”. It
was a Gagnepainia.

At about 1960 our knowledge of the Zingiberaceae was mainly
based on the old ollections of Koenig from Phuket, Johannes Schmidt
from Koh Chang, and the Kerr collections. All in all about 70 species were
documented.

In 1980 I published the first annotated key to the genera of
Zingiberaceae in Thailand. Here the number of species is estimated to ca
150. In 1996 I then published a preliminary list of species and the number
had reached 200. In a newly published book, K. & S.S. Larsen 2005, ’Gingers
of Thailand”, we have documented over 300 species, among which are also
new endemic genera. To some of you the history of these is well known, but
I still find it very important to mention it as it shows how much there is still
to do all over SE Asia.

But, first a word about Caulokaempferia. In 1964 I described the
genus Caulokaempferia based on a group of species formerly treated as
Kaempferia’s. In the following years more species were described and 10
years ago five species from Thailand were known. Today the number has
reached 18. We shall hear even more exiting news about this genus later
today.

Few years ago my friend and collaborator, John D. Mood, came to me
with two specimens that did not match any known taxon. One was collected
in Southern Thailand on the mountains bordering Malaysia. We described
that as Siamanthis siliquosus. Molecular studies have shown that it may
be related to the Bornean genus Burbidgea. Its long silique-like fruits are,
however, similar also to those of Siliquamomum tonkinense from Vietnam.
The other plant Mood brought to me looked like a Kaempferia but with a
yellow Zingiber-like flower. I had indeed myself a colour slide of this plant
given to me years previously, but I had never been able to identify it. It
was a plant that was in the trade in the USA under the name Boesenbergia
aurea, an illegitimate name as it is a later homonym. That, we also described
as a new genus, Cornukaempferia aurantiflora. Shortly afterwards a second
Species turned up and now we have a third species which is about to be

10 Gard. Bull. Singapore 59 (1&2) 2007

published. Molecular studies have shown that it is related to Zingiber. The
third new genus described in the last 5 years is Smithatris. It has an even
stranger history. During an earlier Heliconia meeting in Singapore a plant
was shown that had an inflorescence showing resemblance to a Curcuma
and leaves with the look of a Marantaceae. John Kress and I described it as
Smithatris supraneeana commemorating the late Miss Rosemary Smith, and
at the same time Mrs. Supranee Kongpitchayanond from Thailand, who first
presented it from her nursery, and thus brought it to scientific recognition.
No botanist had ever collected this spectacular plant even though it grows
commonly in a small limestone area just north of Bangkok, an area exploited
by a cement factory. Furthermore it has been used since time immemorial
by the local people to bring to the temples during the celebration of the
Bhuddist Lent, often together with Globba’s. Strangely enough the year
after the Director of the Queen Sirikit Botanical Garden in Chiang Mai, Dr.
Weerachai Nanakorn, had bought some rhizomes at a market in Myanmar
where he was attending a conference, one of these collections turned out
to be a second species, which had almost simultaneously been collected in
the wild by John Kress also in Myanmar. This species was described as S.
myanmarensis. So, three new spectacular new genera described over the
last five years.

But also the number of species in the larger genera found in Thailand
has astonished us. Two genera illustrate this: in my checklist from 10 years
ago I estimated the number of Zingiber species to be ca 25. At that time I
had a Danish Ph D student who undertook a revision of the genus; she came
to the result that the number was 26, but that there were probably two or
three undescribed species. Dr. Ida Theilade, however, became engaged in
a different line of research and handed over her material to Dr. Pramote
Triboun from the Bangkok Herbarium who then started a thorough collecting
programme all over the country for three years. We now know, as we shall
also hear later, that there are over 50 species of Zingiber in Thailand.

A similar result was reached by Dr. Charun Maknoi who has worked
for years on the genus Curcuma where over 10 new species have been
discovered during the last few years, some by John Mood and myself, some
by Professor Puangpen Sirirugsa, others by Dr. Maknoi. Besides all the new
species, many of the species treated as endemic in the Flora of China in the
year 2000 have now been found also to occur in northern Thailand.

So much for the Flora of Thailand, which we regard as one of the
better known regions in tropical Asia. Let me finally add that the genus
Amomum in our checklist in ”’Gingers of Thailand” comes up to 16 species
but according to a team of Thai botanists working on a revision of this genus
the number may be twice as high.

I have tried here in this short overview of the history and the progress

The Exploration of Gingers in SE Asia i

of exploration of the zingiberaceous flora of SE Asia to give you an idea of
how far we have reached and how much we still do not know. And then the
questions come, ” What are the priorities of research in the future? Should
it be molecular studies or should we go out and collect more, or are there
other fields that need to be explored?”

I find that there are three equally important fields:

1. The molecular approach has been shown to be important for a
better understanding of the generic boundaries and relationships
between species and the evolution of the Zingiberaceae as a
whole. It has brought us a big step forward. This is laboratory
work that must be based on a sound knowledge of the identity
of the taxa. And even if there is still much to do it seems to
me that the cream has already been skimmed off the milk.

2. Alpha taxonomy is important and it seems that we are, at the
present time, exploring the last unknown frontiers of SE Asian
biodiversity. New collectors to the region should be aware that
expeditions visiting a tropical country are often taken to the
so-called interesting localities by local botanists. This is where
collectors have grazed repeatedly - and we know from Thailand
that itis outside these areas that all the new discoveries are made.

3. Finally, there is a field that is much neglected: ecological
and biological studies. Pollination and dispersal biology
are unknown in the majority of species, even the fruits and
seeds of many species are unknown. Here local botanists
have a vast research field. But it seems that there is more
prestige in being in the laboratory in a white coat and
working with a big computer than getting out in the jungle.

Is there anything we can do here at this meeting to speed up the
exploration of gingers? As funds are limited it might be a good idea to
establish a kind of advisory board for Zingiberaceae reseach, a group of
experienced local people who could point out areas to which collecting
activities should be directed, taking also regional political aspects into
consideration. Furthermore organizing research groups of young botanists
with a strong scientific leadership that could attract funds. These activities
might be facilitated through a newsletter, probably electronically distributed.
These could be the ways to bring us forward by working together and avoiding
duplication of research. If we can agree here on broad collaboration, to be
open in our research and respect each others work, then, this meeting will
be a big step forward and not only a statement of our knowledge today.

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Gardens’ Bulletin Singapore 59 (1&2): 13-22. 2007 1S

Selected Zingiberaceae Species Exhibiting Inhibitory
Activity Against Mycobacterium tuberculosis H37Rv:
A Phytochemical Profile

A.M. AGUINALDO

Research Center for the Natural Sciences, College of Science, Graduate School
University of Santo Tomas, Espafia, Manila 1023, The Philippines

Abstract

As part of the research efforts to identify plant species which may have
potential against tuberculosis, a study was earlier conducted in collaboration
with the Institute for TB Research, University of Illinois, Chicago, to
randomly screen the crude alcoholic extracts of different plant species using
the MABA assay, to determine any inhibitory activity against the causative
agent, Mycobacterium tuberculosis H37Rv. Of the five species belonging to the
family Zingiberaceae, four were found to inhibit the growth of M. tuberculosis
H;,Rv. These species included Alpinia purpurata (Vieill.) K.Schum., Alpinia
zerumbet (Pers.) B.L.Burtt. & R.M. Sm., Etlingera elatior (Jack) R.M. Sm.
and Zingiber officinale Roscoe. Each species was collected in bulk and
subjected to extraction and several bioassay-directed chromatographic
fractionations. The pure constituents obtained were analyzed for their
structure using spectroscopic techniques. The bioactivity of the pure isolates,
as minimum inhibitory concentration values, was likewise determined. The
results showed the antitubercular activity to be present in the nonpolar
extracts. Structure elucidation of the pure isolates revealed the presence
of sterols (B-sitosterol, stigmasterol), sterol derivatives (B-sitosteryl-B-D-
galactoside, 8-sitosteryl-3-O-6’-palmityl-8-D-glucoside), phenyldecanoids
(6-shogaol and 6-gingerol) and a flavonoid (kumatakenin). Determination
of the MIC showed higher activity of the phenyldecanoids than the steroids,
the steroidal derivatives and the flavonoid.

Introduction

Tuberculosis is a pandemic that has been afflicting the Philippines and other
developing countries. The Philippines is one of six countries with half of all
new cases. The number of reported new TB cases Keeps rising. Because of

14 Gard. Bull. Singapore 59 (1&2) 2007

the rapid increase in TB incidence in Africa, there is a yearly 1% growing
incidence worldwide. Data show that one third of the world’s population
is infected with M. tuberculosis and every year, nearly 2 million deaths are
caused by TB (WHO, 2006a, b).

A number of risk factors affecting TB are HIV/AIDS and multiple
drug resistance. For people living with HIV/AIDS, TB is the single biggest
killer. Latent TB infection is reactivated to an active disease through HIV.
The other risk factor, multiple drug resistance, leads to TB that does not
respond to the standard drug treatment. A WHO survey indicated the
presence of MDR-TB in 109 countries, with the highest rates in China and
former Soviet Union. In 2006, WHO launched a six component “Stop TB
Strategy.” (WHO, 2006b).

Tuberculosis is caused by Mycobacterium tuberculosis (M tb), an
intracellular pathogen affecting higher vertebrates. The search for drugs
against tuberculosis uses the slow growing M tb in its bioassays. In the late
1980’s, the UST Research Center for the Natural Sciences (RCNS) embarked
on a natural products program to screen plants using Mycobacterium 607 or
M. smegmatis, a surrogate fast growing and non-pathogenic organism. After
a number of comparative tests using both organisms failed to show 100%
agreement, it was deemed necessary to screen against the actual etiologic
agent, M. tuberculosis, a unique organism with unique susceptibilities to
drugs.

The Philippines is one of those countries in Southeast Asia with
a rich diversity of flora. Though only 858 species were reported to be
medicinal (Quisumbing, 1978), there are many more plants whose medicinal
properties have not been tested. The general objective of the RCNS TB
Group is to provide a scientific rationalization for the use of plants as
sources of medicine against TB, either for phytopharmaceutical application
or for new drug development. Its specific objectives include the random
screening of plants for inhibitory activity against M. tuberculosis (done
in collaboration with the Institute for TB Research of the University of
Illinois in Chicago), identifying plant families / genera exhibiting inhibitory
activities, isolating through a bioassay-guided procedure the constituents in
the active fractions, identifying the structure of the constituents through a
combination of spectroscopic methods, comparing structural characteristics
and determining the minimum inhibitory concentration of the pure isolate.
This paper collates the results of the different studies done on five species
of Zingiberaceae, namely, Alpinia purpurata (Vieill.) K. Schum., Alpinia
zerumbet (Pers.) B.L.Burtt. & R.M. Sm., Etlingera elatior (Jack) R.M. Sm.,
Hedychium coronarium J. Konig and Zingiber officinale Roscoe (Aguinaldo
et al., 1997; Agbayani et al., 2002; Budoy et al., 2004; Villaflores et al., 2004;
Villaflores, 2005).

Zingiberaceae Species Exhibiting Inhibitory Activity 15

Materials and Methods

Plant material

The voucher specimens were identified and kept at the UST Herbarium
or the National Museum. Table 1 lists the herbarium vouchers, including
locality, collection number, and collector.

Table 1. Herbarium voucher information (location, collection date & number, collector,
herbarium identity).

Location Date of Collection Collector Where deposited
Collection | number

Screening for bioactivity

Approximately 100g plant material (air-dried leaves, fresh rhizomes, fresh
flowers) was ground and extracted with methanol (or ethanol). The filtrate
was concentrated in vacuo at 40 C to give a crude extract. A portion (2
mg) of the crude extract was tested for % inhibitory activity against M.
tuberculosis H3,Rv (Collins and Franzblau, 1997; Fischer et al., 1998). If the
crude extract showed activity, it was partitioned between water and hexane,
dichloromethane and 1-butanol. Each organic layer was concentrated and a
portion similarly tested for bioactivity.

Isolation (bioassay-guided) and structure elucidation

The plant materials (air-dried leaves of A. purpurata, fresh rhizomes of Z.
officinale and E. elatior) were collected in bulk and extracted exhaustively
with alcohol. The crude extract obtained from each was partitioned as
above to obtain the active semicrude extract. The latter was fractionated
repeatedly using silica gel column chromatography with gradient elution
(hexane-DCM, DCM-MeOH) till pure isolates were obtained. Fractions

16 Gard. Bull. Singapore 59 (1&2) 2007

from each chromatographic step were assayed for bioactivity. Isolates were
analyzed using a combination of spectroscopic techniques such as UV, IR,
MS, 'H-NMR and C-NMR, including DEPT, COSY, HMQC and HMBC
These were again assayed for minimum inhibitory concentration (MIC).

Results and Discussion

Table 2 shows the percent inhibitory activity of the crude extracts against M.
tuberculosis H3;Rv at 100 ug/mL. A. purpurata leaves exhibited the highest
activity (94%, 100 ug/mL), followed by E. elatior rhizomes (86%, 100 ug/
mL), and A. zerumbet (80%, 100 ug/mL). Z. officinale showed activity in
the rhizomes but absence of activity in the leaves. Among the five species
of Zingiberaceae tested, only H. coronarium did not exhibit any activity
against M. tuberculosis H3,Rv. Since only the leaves of H. coronarium were
tested and not the rhizomes, it is worth considering the rhizomes for future
tests.

Table 2. Activity of the crude extracts vs. M. tuberculosis H37Rv at 100 ug/mL.

When the crude extracts of A. purpurata rhizomes, leaves and flowers
were tested, the leaves showed the highest activity at two concentrations
(62%, 64 ug/mL; 41%, 32 ug/mL) (Table 3). The percent inhibition values
of the rhizomes and flowers were close (34% and 30%, 64 ug/mL; 21% and
17%, 32 ug/mL) and indicated less activity than the leaves.

Table 3. Activity of the crude extracts from the different plant parts of A. purpurata vs. M.
tuberculosis H37Rv.

Plant part Inhibition (%) Inhibition (%) Inhibition (%)
| ato¢4ug/mL | at 32 ug/mL at 16 ug/mL

Rhizomes | 34 Zh il 12

Leaves 62 41 4

Flowers 30 ig i

Zingiberaceae Species Exhibiting Inhibitory Activity 17

Upon separation of the nonpolar, semipolar and polar constituents
in the crude leaf extract of A. purpurata, the higher activity is in both hexane
and DCM semicrude extracts (64-70%, 64 ug/mL; 58-61%, 32 ug/mL;
34-42%, 16 ug/mL) (Table 4). This indicates that the bioactive constituents
have a nonpolar character.

Table 4. Activity of the semi-crude extracts from A. purpurata leaves vs. M. tuberculosis

H37Rv.
Inhibition (%) Inhibition (%) Inhibition (%)
at 64 ug/mL at 32 ug/mL at 16 ug/mL
Hexane 64 61 34
DCM 70 58 42
n-BuOH 35 10 6

For Z. officinale (Table 5), the bioactive constituents are largely
nonpolar, being present in the hexane semicrude extract (61%, 100 ug/
mL; 19%, 25 ug/mL), with an activity much higher than that in the DCM
(9%, 100 ug/mL) or n-BuOH (-10%, 100ug/mL). Table 5 shows that for the
rhizomes of E. elatior, the active constituents are in the nonpolar (hexane)
and semipolar (DCM) extracts (34% and 35%, respectively, 100 ug/mL).
The data for A. zerumbet show an activity for the DCM extract (76%, 50
ug/mL) which is more than triple that of the hexane extract (23%, 50 ug/
mL). Tables 4 and 5 compare activities of the semicrude extracts per plant
in order to determine the presence of the active constituents. The different
studies used varied concentrations of the semicrude extracts.

Table 5. Activity of the semi-crude extracts from Z. officinale rhizomes, E. elatior rhizomes
and A. zerumbet rhizomes vs. M. tuberculosis H?7Rv.

Z. officinale rhizomes E. elatior rhizomes | A. zerumbet rhizomes
Inhibition (%) Inhibition (%) Inhibition (%) Inhibition (% )
lL at 100 ug/mL at 25 ug/mL at 100 ug/mL at 50 ug/mL
Hexane 61 19 34 23
DCM 9 -4 35 76
n-BuOH -10 -8 0 Not tested

Extracts from the Z. officinale, A. purpurata and E. elatior were
subjected to bioassay-guided isolation till pure isolates were obtained (Fig.
1). The details of the isolation, purification and structure elucidation are
written elsewhere. From Z. officinale rhizomes, the phenyldecanoids 6-

18 Gard. Bull. Singapore 59 (1&2) 2007

shogaol and 6-gingerol were isolated from the hexane extract. The sterols
B-sitosterol and stigmasterol were obtained from the hexane extract of
E. elatior rhizomes. And from the leaves of A. purpurata were obtained
B-sitosteryl-B-D-galactoside, —_-sitosteryl-3-O-6’-palmityl-8-D-glucoside,
and kumatakenin, a flavonoid. The structures were identified upon spectral
analysis and comparison of spectral data with the literature (Zaeoung et
al., 2005; Wright et al., 1978; Gomes and Alegrio, 1998; Shaig et al., 2002;
Urbatsch et al., 1976; Wang et al., 1989).

Upon bioassay of these isolates, the following MICs were obtained:
Z. officinale: 6-shogaol (MIC 64 ug.mL), 6-gingerol (MIC 33 ug/mL); A.
purpurata: %-sitosteryl-B-D-galactoside (MIC >128 ug/mL), B-sitosteryl-3-
O-6’-palmityl-B-D-glucoside (MIC >128 ug/mL), kumatakenin (MIC >128
ug/mL); E. elatior: stigmasterol (MIC >128 ug/mL), B-sitosterol (MIC >128
ug/mL).These results show the higher activity of the phenyldecanoid isolates
from Z. officinale than the sterol glycosides, flavonoid from A. purpurata
and sterols from E. elatior. Furthermore, the high percent inhibitory activity
values of the crude extracts do not necessarily correlate with those of the
pure isolates. With Z. officinale, there was an observed increase in activity
as purification progressed. This was not observed with A. purpurata or E.
elatior where constituents seem to exhibit synergism and are therefore more
active as a mixture.

Having observed a common antitubercular property of extracts from
Zingiberaceae species randomly selected, it is now worth investigating the
other species of Zingiberaceae for reasons of bioactivity targeted search,
and probable taxonomic utilization. With the furtherance of investigations
on equally or more active species, there is sufficient justification for a com-
prehensive phytochemical reexamination of natural products elaborated by
this family.

Acknowledgements

The author thanks Tan Chin Kee Foundation, Dr. Fortunato Sevilla HI (UST
College of Science), Dr. Corazon Menguito (Chemistry Department, UST
CS) for the travel support; Prof. Scott Franzblau for the assays; Prof. Karsten
Krohn (Univ. of Paderborn), Dr. Lindsay Byrne (Univ. of W. Australia), Prof.
Tatsuo Yamauchi (Fukuoka Univ.), Dr. Fumiko Abe (Fukuoka Univ.), for
the spectral analysis; Oliver Villaflores, Christopher Ryan Budoy, Katheryn
Mandap, Roland Marcelo, Metchie Gay Agbayani, Vincent Arbolante, for
the technical data; Asst. Prof. Rosie Madulid (UST Herbarium) and the
National Museum for the botanical identification.

Zingiberaceae Species Exhibiting Inhibitory Activity 19

Figure 1. Compounds isolated from Zingiberaceae species.

CH;0 7 CH30

HO HO

6-shogaol 6-gingerol

R-sitosterol stigmasterol

R-sitosteryl-B-D-galactoside kumatakenin

8-sitosteryl-3-O-6’-palmityl-B-D-glucoside

20 Gard. Bull. Singapore 59 (1&2) 2007

References

Agbayani, M., V. Arbolante, S. Franzblau and A. Aguinaldo. 2002.
Phyanchenieal screening of selected Zingiberaceae species inhibitory to
Mycobacterium tuberculosis H37Rv, p. 28. Abstracts of Papers. 7’ Annual
Convention of the Natural Products Society of the Philippines, United
Laboratories, MetroManila, Natural Products Society of the Philippines.

Aguinaldo, A., M. Bueno, M. Cabanilla, K. Mandap and I. Lapuz. 1997.
The aoa of antitubercular constituents from selected Philippine
plants, pp. 74-76. In: Proceedings of the 13. Annual Philippine Chemistry
Congress, Asiaworld Hotel, Puerto Princesa, Palawan, Philippine
Federation of Chemistry Societies.

Budoy, C., S. Franzblau and A. Aguinaldo. 2004. Steroids from the
antitubercular fraction of the hexane extract of Etlingera elatior
rhizomes, p. 156. Abstracts of Papers, 19 Philippine Chemistry Congress,
Sarabia Manor Convention Center, [loilo City, Philippine Federation of
Chemistry Societies.

Collins, L. and S. Franzblau. 1997. Microplate alamar blue assay vs BACTEC
460 system for high throughput screening of compounds against
Mycobacterium tuberculosis and Mycobacterium avium. Antimicrobial
Agents and Chemotherapy 41: 1004-1009.

Fischer, N. H., T. Lu, C. L. Cantrell, J. Castaneda-Acosta, L. Quiyjano and
». G. Bye ere. 1998. Antimycobacterial evaluation of germacranolides
in honor of Prof. G. H. Neil Towers 75 birthday. Phytochemistry 49:
559-564.

Gomes, D., L. Alegrio. 1998. Acyl steryl glycosides from Pithecellobium
cauliflorium. Phytochemistry 49: 1365-1367.

Quisumbing, E. 1978. Medicinal Plants of the Philippines. Katha Publishing,
JMC Press, Quezon City, Philippines.

Shaig, Ali M., M. Saleem, W. Ahmad, M. Parvez and Y. Raghav. 2002. A
chlorinated monoterpene ketone, acylated B-sitosterol glycosides and
flavanone glycoside from Mentha longifolia (Lamiaceae). Phytochemistry
59: 889-895.

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Urbatsch, L., T. Mabry, M. Miyakado, N. Ohno and H. Yoshioka. 1976.
Flavonol methyl ethers from Ericameria diffusa. Phytochemistry 15:
440-441.

Villaflores, O. 2005. A Flavone and an Acylglucosyl Sterol from the
Dichloromethane Leaf Extract of Alpinia purpurata (Vieill.) K. Schum.
With Inhibitory Activity against Mycobacterium tuberculosis H37Rv. M.
Sc. Chem. Thesis, University of Santo Tomas, Manila.

Villaflores, O., A. Macabeo, D. Gehle, K. Krohn, S. Franzblau and A.
Aguinaldo. 2004. A flavonoid from Alpinia purpurata (Vieill.) K. Schum.,
pp. 34-35. Abstracts of Papers. 9th Annual Convention of the Natural
Products Society of the Philippines, U.P. Diliman, Quezon City, Natural
Products Society of the Philippines.

Wang, Y., M. Hamburger, J. Gueho and K. Hostettman. 1989. Antimicrobial
flavonoids from Psiadia trinervia and their methylated and acylated
derivatives. Phytochemistry 28: 2323-2327.

World Health Organization. 2006a. Tuberculosis Facts. Retrieved June 26,
2006 from the World Wide web: http:/www.who.int/tb/publications/en/

World Health Organization. 2006b. Tuberculosis, pp 1-4. Retrieved June
26, 2006 from the World Wide Web: http.//www.who.int/mediacentre.

factsheets/fs104/en/index.html

Wright, J. L.C.,A.G. McInnes, S. Shimizu, D. G. Smith, J. A. Walter, D. Idler
and MW. Khalil. 1978. Identification of C-24 alkyl epimers of marine sterols
by “C nuclear magnetic resonance spectroscopy. Canadian Journal of
Chemistry 56: 1898-1903.

Zaeoung, S., A. Plubrukarn and N. Kewapradub. 2005. Cytotoxic and free
radical scavenging activities of Zingiberaceous rhizomes. Songklanakarin
Journal of Science and Technology 27: 799.

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Gardens’ Bulletin Singapore 59 (1&2): 23-34. 2007 23

Ethnobotanical Notes on Gingers of the Huon Peninsula
in Papua New Guinea

B.B. BAU’ AND A.D. POULSEN’

‘Papua New Guinea Forest Research Institute, PO. Box 314, Lae 411 Morobe
Province, Papua New Guinea
“Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, Scotland

Abstract

Only few studies on useful gingers in Papua New Guinea have been published
and we were only able to find information on two commonly used species. We
conducted a 2-weeks preliminary study in the Huon Peninsula to document
the species of gingers and their local names in the Kote language and their
uses by the indigenous people. All species encountered were useful: four
species of Etlingera, three species of Amomum and one species of Zingiber.
It is recommended that further surveys should be conducted on gingers
of Papua New Guinea to understand their taxonomy and ethnobotany in
order to devise an appropriate ginger conservation program for the local
communities.

Introduction

Papuasia, including Papua New Guinea (PNG), harbours approximately
8 native genera and 207 species of gingers (Zingiberaceae) as proposed
by Hoft (1992). The family still needs to be thoroughly surveyed before
the total species pool can be determined. With more than 800 language
groups, PNG is at the same time culturally very diverse. Most of these
groups have developed intricate people-to-plant associations including
local names and uses for many plants. The uses of plants are still very
important for the subsistence strategies employed by the people in many
remote villages (Damas, 1998). Detailed ethnobotanical studies in Borneo
have found that the ginger family (Zingiberaceae) includes numerous
species useful to the local people (Christensen, 2002) and many of these
may also be horticulturally important. It is likely that the people of PNG
still maintain comprehensive ethnobotanical knowledge but it needs proper
documentation as soon as possible before the information disappears

24 Gard. Bull. Singapore 59 (1&2) 2007

due to the increasing globalisation. The information on gingers and their
traditional uses will be important in order to facilitate proper management
and conservation strategies either in situ or ex situ.

Several papers and books have already been published on
traditionally important plants of Papua New Guinea as medicines but had
made less emphasis on other uses (Paijmans, 1976; Woodley, 1991). Only
two gingers have so far been reported as being commonly used: Amomum
aculeatum Roxb. and Zingiber officinale Roscoe (Holdsworth and Mahana,
1983), both of which are used against fever or influenza. The latter is also
used throughout Papua New Guinea by the indigenous people to relieve
cough (Holdsworth, 1977; Holdsworth and Damas, 1986).

Study Area

The study was conducted in November 2001 inland from Finschhafen on
the Huon Peninsula at Jivevaneng Village (6°30’S 147°47’E) at 300-500
m above sea level and at Nanduo Village (6°26’S 147°40’E) at 600-700 m
above sea level (Fig. 1.). Both of these villages are inhabited by people
speaking the Kote language and have a patrilineal society (R. Banka, 2001,
pers. comm.). The Finschhafen area has many coastal and inland villages
that are separated by the rugged terrains comprising the Cromwell and the
Saruwaged Ranges. The geological aspects of the topography are a major
factor that makes the villages inaccessible to basic governmental services.
Because of inaccessibility the people still rely largely on forest resources for
their survival. The habitat surrounding the village includes old garden sites,
and the degraded roadside areas are composed of common weeds species.
More pristine forest is found a few kilometres away from the villages.

Materials and Method

The following standard methods were used for plant collecting. The vegetation
was explored to locate the conspicuous leafy shoots of ginger. The search was
subsequently intensified to find flowers and fruits. Photographs were taken
of these before the rhizomes were dug up. This was done carefully to avoid
breaking any attached inflorescences or infructescences. The specimens
were collected in three sections: 1) the base with the rhizomes and flowers
or fruits attached (if any), 2) the mid-section with 2-3 leaves, and 3) the top
of the shoot with 4-5 leaves depending on size of leaf.

Interviews with guides from the nearby villages were conducted
during the collecting and notes were taken on local names in the Kote
language and uses. The collections were deposited at the Papua New Guinea

Ethnobotanical Notes on Gingers of the Huon Peninsula 2D

New ireland Group

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Figure 1. The study sites are situated approximately 100 km E of Lae in Morobe Province,
Papua New Guinea. (Map courtesy Encarta Encyclopaedia of 1988-1997).

National Herbarium (LAE) with duplicates sent to the Royal Botanic
Gardens, Kew (K) and University of Aarhus (AAU), Denmark.

Results

All collected species (eight) of gingers were found to have known uses by
the Kote speaking villagers at the Huon Peninsula: four species from the
genus Etlingera; three species from the genus Amomum, and one species
from the genus Zingiber. Each species has one to four uses. The information
from seven of these species is documented for the first time in the present
paper, whereas the information on one species has already been published.

Description of species

Amomum aculeatum Roxb.
Material collected: Bau et al. LAE 86303, Jivevaneng, 9 Nov 2001 (LAE).
Vernacular name: Asabareng

26 Gard. Bull. Singapore 59 (1&2) 2007

Uses: 1) The pseudostem of young leafy shoot is beaten and the extracted
juice is rubbed externally on the body and head and has a cooling effect in
easing pain. 2) The fruits have a sweet taste and are eaten.

Description: Terrestrial herb to 2.5 m; rhizome thick; leafy shoots to 20 cm
apart; base to 5 cm diam.; sheath brown at base - green at top, glabrous and
shiny; ligule slightly bilobed; petiole 1.5 cm, base slightly purple; lamina obo-
vate, plain green, pale green beneath, glabrous, midrib pale or white, apex
acuminate, base + oblique. Inflorescence radical, peduncle to 10 cm long;
corolla lobes pale pink; labellum white with red lines in centre basally and
yellow above; stamen white, anther crest with two extended wings. Infruct-
escence globular; fruits dark purple, indehiscent; lobes extended above, with
yellow lines to base. Fig. 2.

Habitat: ridge top near an old garden near village.

Figure 2. Inflorescence of Amomum aculeatum Roxb. This species has very juicy fruits when
ripe and they are edible. Photo: A.D. Poulsen

Amomum maximum Roxb.

Material collected: Bau et al. LAE 86308, Jivevaneng. 9 Nov 2001 (LAE).
Vernacular name: Sdsiric

Uses: 1) The inner part of the young leafy shoot is scraped and applied
externally on the knee against aches. 2) The fruits are eaten by bandicoots
and therefore the plant is grown as an attractant to facilitate hunting near
the village.

Description: Terrestrial herb to 2 m; leafy shoots clumped; leafless to 1.3
m, leaves clustered at top, 8-10 leaves per shoot; base of leafy shoot 5 cm
diam. gradually decreasing to 2 cm; sheath yellowish at base, pale green to
the top; ligule membranous, bilobed, caducous; petiole 5 cm; lamina plicate,

Ethnobotanical Notes on Gingers of the Huon Peninsula On|

plain green,.dull pale beneath, pubescent, almost corrugated, base oblique.
Inflorescence radical from rhizome; peduncle 6—11 cm long, bractless, pale
green, bracts in spike not persistent. Flowers not seen. Infructescense,
globular; fruits irregularly winged, immature, yellow-green, remnants of
persistent calyx. Fig. 3.

Habitat: Common in old gardens near village at about 300 m elevation.

Figure 3. Amomum maximum Roxb. The fruits attract the bandicoots which is a desired
game species. Thus the A. maximum is planted to make hunting easier. Photo: A.D. Poulsen

Amomum sp. 1

Material collected: Bau et al. LAE 86358, Nanduo, 14 Nov 2001 (LAE).
Vernacular name: Bareng-bafu

Uses: The flesh inside the leafy shoot is scraped off, squeezed, strained and
drunk to treat colds, flu, stomachache and headache.

Description: Terrestrial herb, 2.6—3.8 m tall, leafy shoot with 10-28 leaves,
clumped; base of leafy shoot white; sheath green, glabrous, ligule bilobed
pale green; petiole short; lamina to 45 x 8.5 cm, green, glabrous above,
beneath pale green. Inflorescence radical, peduncle 10-12 cm long; calyx
brown, persistent; corolla lobes pale yellow; labellum tunnel-shaped, yellow
with red stripes forming into the corolla, apical lobe shorter than style;
anther crest white. Infructescence green, immature seeds white. Fig. 4.
Habitat: Disturbed forest area near village.

28 Gard. Bull. Singapore 59 (1&2) 2007

Figure 4. Amomum sp. 1, rhizomes, fruits, flowers and leaves. The flesh of the leafy shoots are
used to treat colds and flu, stomach ache and body ache. Photo: A.D. Poulsen

Etlingera labellosa (K. Schum.) R.M. Sm.

Material collected: Bau et al. LAE 86301, Jivevaneng, 9 Nov 2001 (LAE).
Vernacular name: Barengopo

Uses: 1) Leaves are used to cover tubers of Colocasia antiquorum Schott
when these are boiled in water with added oil to prepare a meal. 2) The
beaten pseudostem (using hard sticks) is squeezed to extract juice, which is
applied externally against body ache. 3) The twisted and beaten pseudostem
is used for making climbing ropes. 4) Fruits are sweet and eaten.
Description: Terrestrial herb to 4 m; rhizome long-creeping; unpleasant smell
of cabbage and soap when cut; scales on rhizome white or greenish brown
when exposed; base of leafy shoot to 8 cm diam.; sheath green and glabrous;
petiole 1.5-2.5 cm, green; lamina to 78 x 15 cm, plain green beneath; base
unequal. Inflorescence radical from rhizome; peduncle 3—7 cm long, pale
brown to white; calyx white to pale pink at apex; central lobe of labellum
emarginate, lateral lobes pale red to white; filament white, anther white with
central pink and pink at crest; stigma dark pink. Infructescence subterranean,
with 1-2 fruits; fruit ca 2.5—-3 cm diameter; densely covered by pale brown
hairs. Fig. 5.

Habitat: Near forest trail in an old garden area near village.

Ethnobotanical Notes on Gingers of the Huon Peninsula 29

Figure 5. The leafy shoots of Etlingera labellosa (K. Schum.) R.M. Sm. are used as climbing
ropes for coconut trees. The flesh is also used to treat all kinds of body aches. Photo: A.D.
Poulsen

Etlingera sp.1

Material collected: Bau et al. LAE 86300, Jivevaneng, 9 Nov 2001 (LAE).
Vernacular name: Safanang

Uses: The leaves are covered with the bark of Paraserianthes falcataria (L.)
Nielsen and cooked over the fire; the cooked leaves produce a very strong
aromatic smell and are worn as traditional decorations during sing sings
(traditional dances) and folk celebrations.

Description: Terrestrial herb to 1.5 m; rhizome long-creeping, when cut with
strong taste and smell of anis seed (Pimpinella anisum L.); base of leafy
shoot to 2 cm diameter; sheath green, reticulate, not pubescent; ligule at least
5 mm, green; lamina plain green, young leaves reddish brown. Inflorescence
from rhizome some distance from base; peduncle 2—8 cm long; bracts pale
pink, calyx pale red, petals dark pinkish red; labellum pink; stamen pale
pink; stigma white.

Habitat: The population was found near a bush track in an old garden area. It
was growing amongst Bambusa sp. below several cultivated Cocos nucifera
Ce

Etlingera sp. 2

Material collected: Bau et al. LAE 86302, Jivevaneng, 9 Nov 2001 (LAE).
Vernacular name: Gamiong

Uses: The fruits are chewed as a substitute for betel nut (Areca catechu L.).
Description: Terrestrial herb to 2.5 m; rhizome + long-creeping, scales brown;

30 Gard. Bull. Singapore 59 (1&2) 2007

leafy shoot with c. 20 leaves; base of leafy shoot to 3 cm diam., brownish
green; sheath reticulate, greenish brown, pubescent on cross-ribs; petiole 0.5
cm; lamina plain green. Young inflorescence pink, flower plain red, anther
brown, stigma white. Smell of cut rhizome and crushed leaves faintly like
Etlingera elatior (Jack) R.M. Sm.

Habitat: Old garden site near village.

Etlingera sp. 3

Material collected: Bau et al. LAE 86337, Bembavaneng Hill, Nanduo, 14
Nov 2001 (LAE).

Vernacular name: Zunzun

Uses: 1) The fruit is chewed as betel nut (Areca catechu L.).2) The stems and
leaves are used to make small shelters to hide in when hunting bush fowls.
Description: Terrestrial herb 1—2 m; diameter 3 cm, rhizome 1 cm diameter,
long creeping; scales reddish, sheath olive-green, reticulate; sheath purple
at top; leaves plain green, glabrous above, pubescent below, young purplish
brown below, inflorescence less than 5 cm long; flowers plain red; anther
pale pink; stigma white.

Habitat: Forest near village.

Zingiber zerumbet (L.) Sm.

Material collected: Bau et al. LAE 86304, Jivevaneng, 9 Nov 2001 (LAE).
Vernacular name: Zazamang

Uses: Ornamental plant in village and garden areas.

Description: Terrestrial herb to 1 m;rhizome thick and short, yellow in centre
when cut; base of leafy shoot fleshy, reddish, to 2 cm; sheath purplish; ligule
to 2 cm, membranous; petiole swollen; lamina plain green, soft, beneath pale
green and puberulent. Inflorescence radical, to 20 cm long; bracts reddish.
Flowers not seen.

Habitat: In a cluster below several planted Cocos nucifera L. trees along
trail in village garden.

Discussion

The total species pool of the study area is no doubt larger than the eight
species collected in the present study, but a more intensive survey would
need to be conducted to document more species. This should include more
focus on the dominant species in the old garden sites, which were poorly
covered in our preliminary survey. Likewise, some common cultivated
species like Zingiber officinale Roscoe were not sighted as the survey took
place away from the most likely areas of its cultivation.

Ethnobotanical Notes on Gingers of the Huon Peninsula OL

The uses of gingers in Papua New Guinea have, to date, been poorly
documented, and the results of this preliminary survey indicates that the
species have a wide range of uses from ethnic dressings in traditional dances
(sing sings) to an alternative for betel nut chewing, and thus have a large
potential.

Previous publications only highlighted the genera Amomum and
Zingiber as useful to the villagers in the Huon Peninsula (Holdworth, 1977;
Holdsworth and Damas, 1986; Holdsworth and Mahana, 1983; Paijmans,
1975; Peekel, 1984; Woodley, 1991). But this study also documents uses for
the genus Etlingera which was found to have several uses. In the present
paper, we are only able to give specific epithets for one species, because
Etlingera is still poorly known in PNG and is still being revised for the Flora
Malesiana by the second author.

In the present study, Zingiber zerumbet (L.) Sm. is only used as an
ornamental plant but in Malaysia it is also used as medicine (Holttum, 1950;
Burkill, 1966). In the Bismarck Archipelago, however, Peekel (1984) noted
it to be ‘less spicy’ than Z. caninum Peekel and Z. foliatum Peekel but no
specific notes of its edibility was presented.

Most of the useful species are common around the home gardens,
which means that they are either cultivated to some degree or associated
with the secondary forest vegetation. Also, in Borneo many gingers thrive
in disturbed or human influenced vegetation (Christensen, 2002; Poulsen,
2006).

A more extensive survey of the gingers of Papua New Guinea
commenced in Dec 2006 during which additional areas were visited to
obtain more collections and ethnobotanical information. This will provide
an essential basis for a comprehensive systematic treatment. Combined with
ethnobotanical information this may be used to identify endangered and/or
endemic species where particular conservation efforts have to be conducted.
This will eventually contribute towards a more meaningful and constructive
conservation avenue whether it is in-situ or ex-situ. Such a recommended
conservation program should be community-based and economically
attractive for the local villagers. The conservation program should be
a source of income for the local people who will then be encouraged to
propagate the wild seedlings in smaller nurseries for potential horticultural
uses nationally and internationally.

Acknowledgements

The authors thank the Organizing Committee of the 4" International
Symposium on the Family Zingiberaceae and the Papua New Guinea Forest

32 Gard. Bull. Singapore 59 (1&2) 2007

Research Institute for providing funding for their participation, and for the
first author to present this paper, at the ginger symposium in Singapore in
2006. We are also grateful to Mr. Roy Banka for the logistics and technical
advice provided while conducting the survey, the Papua New Guinea Forest
Research Institute for the use of the facilities, the councillors and the people
of Jivevaneng and Nanduo villages for their support and sharing of their
knowledge.

References

Burkill, I.H. 1966. A Dictionary of the Economic Products of the Malay
Peninsula. Ministry of Agriculture, Kuala Lumpur. Malaysia.

Christensen, H. 2002. The Ethnobotany of the Iban & the Kelabit. - A joint
publication by Forest Department of Sarawak, Malaysia, Nepcon,
Denmark & The University of Aarhus, Denmark.

Damas, K. 1998. The present status of plant conservation in Papua New
Guinea. Rare, threatened and endangered Floras of Asia and the Pacific
Rim, Academia Sinica Monograph Series 16: 171-179.

Holdsworth, D. 1977. Medicinal plants of Papua New Guinea. Noumea,
South Pacific Commission.

Holdsworth, D. and K. Damas. 1986. Medicinal plants of Morobe Province,
Papua New Guinea. Part III. The Finschhafen Coast. /nternational
Journal of Crude Drug Research 24: 217-225.

Holdsworth, D. and P. Mahana. 1983. Traditional medicinal plants of the
Huon Peninsula, Morobe Province, Papua New Guinea. /nternational
Journal of Crude Drug Research 21: 121-133.

Holttum, R.E. 1950. The Zingiberaceae of the Malay Peninsula. Gardens’
Bulletin Singapore 13: 1-249.

Hoft, R. 1992. Plants of Papua New Guinea and the Solomon Islands -
Dictionary of the Genera and Families of Flowering Plants and Ferns.
Wau Ecology Institute Handbook No. 13. Wau, Papua New Guinea.

Paijmans, K. (ed.) 1995. New Guinea Vegetation. Australian National
University Press, Canberra, Australia.

Ethnobotanical Notes on Gingers of the Huon Peninsula 35

Peekel, P.G. 1984. Flora of the Bismarck Archipelago for Naturalists. Division
of Botany, Lae, Papua New Guinea.

Poulsen, A.D. 2006. Etlingera of Borneo. Natural History Publications
(Borneo). 263 pp.

Woodley, E. (ed.) 1991. Medicinal Plants of Papua New Guinea; Part 1:
Morobe Province. Wau Ecology Handbook No. 11, Verl. Margraf,
Weikersheim.

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Gardens’ Bulletin Singapore 59 (1&2): 35-40. 2007 35

Rapid In Vitro Propagation of Hornstedtia reticulata
(K. Schum.) K. Schum.

HLH. BAY ', H.B. SANI',P.C. BOYCE’ AND S.L. SIM |

‘Institute of Biodiversity and Environmental Conservation, Universiti Malaysia
: Sarawak 94300 Kota Samarahan, Sarawak, Malaysia
Malesiana Tropicals, suite 9-04, Tun Jugah Tower, No.18 JIn Tunku Abd Rahman,
90100, Kuching, Sarawak, Malaysia
* Email of corresponding author: bay_ivy@yahoo.com

Abstract

Seeds of Hornstedtia reticulata (K. Schum.) K. Schum. collected from the
wild were double surface sterilised with 30% Clorox, followed by 15%
Clorox, each for 20 minutes. The sterilised seeds were sown on Gamborg B5
medium. The meristems of 12 weeks old seedlings, including the basal parts
of leaf sheath, were used to induce multiple shoots in Gamborg B5 media
incorporated with 6-benzylaminopurine (BAP) alone (2mg/L and 3mg/L)
and in combination with o-naphthalena acetic acid (NAA) at different
concentrations (0.5mg/L and 0.1mg/L). Observation showed that all the
treatments were able to produce multiple shoots while the highest number
of shoots was obtained from explants that were treated with 3mg/L BAP
after three subcultures.

Introduction

Hornstedtia Retz. is a well-defined genus characterized by the rigid
involucre of sterile bracts, which encloses the entire inflorescence from the
uppermost part of the open flowers. Valeton (Bull. Jard. Bot Buitenz. Ser.
3, 3: 150-179, 1921) cited by Smith (1985) had subdivided Hornstedtia into
three subgenera, Hornstedtia, Elettariostemon and Rosianthus. The Bornean
plants all fall within the first two groups.

Hornstedtia reticulata (K. Schum.) K. Schum. is very distinctive. The
cyathiform inflorescence is borne on stilt roots, and the sterile bracts, which
are the most strongly reticulated of all Bornean Hornstedtia, are scabrid
to the touch. Study on species of Hornstedtia is scarce; hence, information
beyond taxonomy study is still unavailable.

Up to this moment, several species of Zingiberaceae with established

36 Gard. Bull. Singapore 59 (1&2) 2007

uses as condiments, spices and as ornamental plants have been investigated
for in vitro multiplication (Borthakur et al., 1999; Jasrai et al.,2000; Salvi et al.,
2000; Rout et al., 2001; Khatun et al., 2003; Prakash et al., 2004; Wondyifraw
et al.,2004). So far, no successful micropropagation protocol for Hornstedtia
spp. has been reported. For this reason, this study served as a preliminary
research on in vitro propagation of Hornstedtia reticulata to mass produce
genetically uniform plantlets for future conservation plan and ornamental
purpose.

Materials and Methods

Materials
Seeds of Hornstedtia reticulata were collected at Kampung Segong, Bau,
Kuching, Sarawak, Malaysia.

Methods

(a) Media preparation

The culture media Gamborg B5 was used in the study (Gamborg et al.,
1968). The medium contains 30g/L sucrose and vitamins. After adjusting
to pH 5.8+1, medium was solidified using 3g/L gelrite. Culture media were
sterilised by autoclaving at 104kPa at 121°C for 20 minutes.

(b) Surface sterilisation and sowing of seed

The seeds were soaked in distilled water overnight to remove the mucilage
layers. After that, the seeds were soaked in 75% ethanol for one minute
before double surface sterilised with 30% Clorox, followed by 15% Clorox,
each for 20 minutes. They were then rinsed three times in sterilised distilled
water before cultured in B5 media incorporated with 5ml/L PPM and
Smg/L Tetracyclin for seven days. After seven days, the seeds were sown on
Gamborg B5 medium

After 12 weeks of culture, 30 seedlings, each 4-5 cm in height, were randomly
selected for study on the effects of 6-benzylaminopurine (BAP) alone or
with the combination of o-naphthalene acetic acid (NAA) in different
concentration. Three replicates were used for each treatment. The roots, the
leaves, and the leaf sheaths of seedlings were removed. Then, the meristems
of 12 weeks old seedlings, which were cut into approximately | cm in length,
were used to induce multiple shoots. The explants were monthly subcultured
in fresh media with growth regulators incorporated for the first two months
and thereafter in the plant growth regulator-free medium. Observation on

Rapid in Vitro Propagation of Hornstedtia reticulata BT

the number of explants forming shoots was recorded. Data were subjected
to factorial analysis using General Linear Model.

Figures A - B2. Shoot multiplication of Hornstedtia reticulata. A. A clump of adventitious
shoot buds with’ primordia excised from meristem explants after 6 weeks of subculturing in
media with 3mg/l BAP; B1. A clump of adventitious shoot buds from the basal potion of
meristem region in media supplemented with 3mg/l BAP + 0.1mg/l NAA after 8 weeks of
subculturing; B2. The clump was cut into two halves and both continued to form cluster of
shoots after subcultured onto basal medium.

Results and Discussion

All the treatments having BAP alone, or with the combination of NAA in
different concentrations, were able to generate multiple shoots. However,
the rate of bud multiplication was significantly different according to the
BAP + NAA formulations. Based on Fig. 1, number of shoots produced
from explants in 3mg/l BAP was significantly different from the explants

38 Gard. Bull. Singapore 59 (1&2) 2007

cultured in 2mg/l BAP. Frequency of shoot proliferation was highest at
3mg/L BAP alone. The average number of shoots was 9.67 + 2.31. Numerous
adventitious shoots were observed near the basal potion of the shoot cluster
after 12 weeks of subculturing. Multiplication rate in media incorporated
with 2mg/L BAP was lowest among the treatments with the mean reading
of 4.33 + 2.31.

In this study, addition of NAA into medium with BAP was proven not
efficient in increasing number of multiple shoots. Similar effect was showed
in Zingiber petiolatum, where maximum shoots regeneration from terminal
buds explants was obtained on medium with 4.4 uM BAP alone, while lesser
shoots were obtained from the explants that cultured in the media with ad-
dition of 0.5 uM NAA (Prathanturarug et al., 2004). Furthermore, similar
result was also encountered when the apical meristems of Curcuma amada
Roxb. and Zingiber officinale Rosc. were explanted on media supplemented
with BAP alone and BAP incorporated with NAA where the percentage of
bud growth were decreased 50% and 67% respectively (Jasrai et al., 2000).
Hence, addition of NAA is not recommended for most of the Zingiberaceae
plant species.

Observation also illustrated that the explants were started to show
significantly increase in number of shoots produced after they were subcul-
tured onto basal medium without PGR. The same finding was found in oth-
er Zingiberaceae plant species, such as Curcuma longa L. and Zingiber peti-
olatum (Prathanturarug et al., 2003, 2004). Medium for induction of rooting
of shoots was not required as the regenerated plantlets produced plentiful
roots in the same growth regulators media. Similar observation was seen
in plantlets produced from shoot tip explants of Zingiber officinale Rosc.
in MS media supplemented with BAP + Kinetin treatments (Khatun et al.,
2003).

f shoots

b> @
> |
Fez
| = | z
=
eeu NAA
© | oa
= | O omg/I NAA
oO

0.1mg/I NAA

O 0.5mg/ NAA
2mg/| BAP 3mg/l BAP

Figure 1. Marginal Means for number of shoots produced.

Rapid in Vitro Propagation of Hornstedtia reticulata 39

Conclusion

This report had demonstrated the preliminary result of using tissue culture
method as a possible mean for producing large number of true-to-type
plantlets of Hornstedtia reticulata. BAP alone in concentration of 3mg/I is
sufficient enough to micropropagate the plantlets which can be exploited
for further research of this species. Currently, the acclimatization has not
yet been performed because the regenerated plantlets are kept for further
molecular study. Hence, acclimatization will be performed later.

Acknowledgements

The authors would like to acknowledge the IGS fund (Grant no. IGS
(R&D) 16/03) granted by The Ministry of Science, Technology and Innova-
tion (MOSTI), Malaysia, for financial support of the research and the first
author (H.H. Bay) would like to thank MOSTI for awarding her the MOS-
TI scholarship.

References

Borthakur, M., J. Hazarika and R.S. Singh. 1999. A_ protocol for
micropropagation of Alpina galanga. Plant Cell, Tissue and Organ
Culture 55: 231-233.

Gamborg, O.L., R.A. Miller and L. Ojima.1968. Nutrient requirements of
suspension culture of soybean cells. Experimental Cell Research 50: 150-
£58.

Jasrai, Y.T., K.G. Patel and M.M. George. 2000. Micropropagation of Zingiber
officinale Rosc. and Curcuma amada Roxb. Centennial Conference on
spices and Aromatic Plants 1: 52-54.

Khatun, A., S. Nasrin and T.M. Hossain. 2003. Large scale multiplication of
ginger (Zingiber officinale Rosc.) from shoot-tip culture. Online Journal
of Biological Sciences 3: 59-64.

Salvi D., L. George and S. Eapen. 2000. Direct regeneration of shoots from
immature inflorescence cultures of turmeric. Plant Cell, Tissue and Organ
Culture 62: 235-238.

40 Gard. Bull. Singapore 59 (1&2) 2007

Prakash, S., R. Elangomathavan, S. Seshadri, K. Kathiravan and S.
Ignacimuthu. 2004. Efficient regeneration of Curcuma amada Roxb.
plantlets from rhizome and leaf sheath explants. Plant Cell, Tissue and
Organ Culture 78: 159-165.

Prathanturarug S., N. Soonthornchareonnon, W. Chuakul, Y. Phaidee and P.
Saralamp. 2003. High-frequency shoot multiplication in Curcuma longa
L. using thidiazuron. Plant Cell Reports 21: 1054-1059.

Prathanturarug S., D. Angsumalee, N. Pongsiri, S. Suwacharangoon and T.
Jenjittikul. 2004. In vitro propagation of Zingiber petiolatum (Holtum)
I. Theilade., a rare Zingiberaceous plant from Thailand. Jn Vitro Cell
Development Biology-Plant 10: 317-320.

Rout, G.R., S.K. Palai, S. Samantaray and P. Das. 2001. Effect of growth
regulator and culture conditions on shoot multiplication and rhizome
formation in ginger (Zingiber officinale Rosc.) in vitro. In Vitro Cell
Development Biology - Plant 37: 814-819.

Smith, R.M, 1985. A review of Bornean Zingiberaceae: I (Alpineae p.p.).
Notes from the Royal Botanic Gardens Edinburgh 42: 261-314.

Wondyifraw, T. and S. Wannakrairoj. 2004. Micropropagation of Krawan
(Amomum krervanh Pierre ex Gagnep). Science Asia 30: 9-15.

Gardens’ Bulletin Singapore 59 (1&2): 41-46. 2007 41

Variations in Tissue Development and Secondary Product
Elaboration of Hedychium coronarium J. Konig Floral
Cultures Grown on Different Media

L.B. CARDENAS

Institute of Biological Sciences, University of the Philippines Los Banos,
College, Laguna, Philippines 4031

Abstract

The studies on the variations in tissue development and secondary
productelaboration of Hedychium coronarium J. K6nig, locally known as
camia, in culture on different growth media, using the floral tube part, are
reported.

- Introduction

The use of biotechnology in the study and harvest of important natural
products is a valued approach especially now that the natural resources are
fast dwindling. Fascination for plant essential oils, for instance, dates back to
antiquity and their production in plant tissue cultures has long been sought
for. However, experience shows that not all natural products produced by
the plant could be synthesized in its unorganized tissue culture or callus. Most
times, tissue differentiation is a requirement. Previous work on essential
oils obtained from Apiaceae tissue culture indicated that some components
of the essential oil like the phenylpropanoids can be synthesized in the
callus but not the mono- nor the sesquiterpenes (Cardenas, 1993). Thus, the
essence produced in tissue culture can only approximate the full scent of the
plant depending partly on the degree of tissue differentiation attained.

“Camia”, Hedychium coronarium J. Konig of the Zingiberaceae,
carries short-lived, white, sweet scented flowers. A study on the tissue
culture of camia flowers was attempted to check on any natural products
it can synthesize and store. A growth medium previously proven to sustain
alkaloid production in Catharanthus roseus (L.) G. Don floral tissue culture
was used (Cardenas, 1983).

42 Gard. Bull. Singapore 59 (1&2) 2007

Materials and Methods

Floral tube of yet unopened camia flowers was used as explant for the
experiment. Whole unopened flower buds were surface sterilized in 3%
CaOCl aqueous solution for 5 mins. The floral tube was excised after three
consecutive rinsing in sterile distilled water and cut into 1-mm sections. These
were inoculated into two different growth media (Table 1). The first was
“MS” Murashige and Skoog medium (Murashige and Skoog, 1962) and the
second “mWB” was a modification of the Wood and Braun medium (Braun
and Wood, 1962). The two media differed only in their macroelements.
Their microelement and vitamin composition are similar following those
recommended for MS medium. Both media were supplemented with 3 ppm
NAA (naphthalene acetic acid) and 0.5 ppm Ki (kinetin), 3% sucrose and
0.2% Gelrite. The pH was adjusted to 5.8 prior to autoclaving.

The cultures were maintained at ambient room temperature of 28°C
with 8-hr daylight provided by a west- -facing window. Low diffused light,
with the highest value at 5.20 umolS” m recorded in the early afternoon, was
observed. Observation on the basic anatomy of the growing callus was made
after 6 weeks of culture under the light microscope. The orange pigment
produced by the callus was extracted with absolute methanol in the dark
and the absorption spectra in visible light (380-780 nm) determined using
Labomed” spectro dual split beam UV-VIS spectrophotometer, USA.

Table 1. Comparison of the MS and mWB growth media used in the experiment.

MS (Physiol. Plant. 18: 100) in mg/l: mWB (PNAS 48: 1776) in mg/I:

370 MgSOs-7H20 MegSOs-7H2O
NazSOs

440) CaCh-2H20 Ca(NOs)2

170 KH2PO. KCl
NaH2POs

1900 KNOs KNOs
NaNOs

1650 NHsNOs3 (NH3)2SOs

Variations in Tissue Development and Product Elaboration of Hedychium coronarium 43

Results and Discussion

The floral tube segments produced callus slowly in two months from
inoculation with the 3ppm NAA and 0.5 ppm Ki supplements. Unexpectedly,
though, the white explant on mWB changed to orange in color and this
color was maintained in the callus that developed. Succeeding subcultures
of the callus to media of the same composition proved that the pigment was,
indeed, synthesized and sequestered in all cells of the tissue culture. Seldom
is chlorophyll produced (Fig. 1). In contrast, MS-grown callus remained
mostly unpigmented, typical of cultures from unpigmented explants. As the
latter matured, roots were initiated (Fig. 2).

For the Zingiberaceae,the most studied pigments are the curcuminoids:
curcumin, monodemethoxycurcumin and bisdemethoxycurcumin of the
Curcuma species. These are cinnamoyl pigments produced and stored in
the rhizome with recorded biological activities, particularly for curcumin
(Wagner and Bladt, 1996). On the other hand, no pigment analysis for H.
coronarium was encountered. Fruit set among the local populations elsewhere
is apparently low, unlike the plants of H. coronarium at the Singapore
Botanic Gardens that produced big orange fruits during the time of the 4"
International Symposium on the Zingiberaceae in July 2006. The fruit of the
local species populations in the Philippines, encountered only once by this
researcher in October 2006, is small and hidden inside the floral bract.

The orange color of fruits is mostly attributed to carotenoids and
there are over 600 natural carotenoids known, including those in algae,
fungi and bacteria (Rodriguez-Amaya, 1999). It can not be discounted that
the orange pigment(s) synthesized in camia callus is of the carotenoids.

Figure 1. H. coronarium callus from floral tube explant two months after inoculation on
mWB medium supplemented with 3ppm NAA and 0.5 ppm Ki.

44 Gard. Bull. Singapore 59 (1&2) 2007

Figure 2. H. coronarium callus from floral tube explant two months after inoculation on MS
medium supplemented with 3ppm NAA and 0.5 ppm Ki.

Anatomical observations of the callus showed some short trichomes
that might be a carry over from the explant. Unorganized callus growth was
evident. Compared with carrot and squash that are well studied for their
carotenoids, the camia callus methanol extract exhibited a different TLC (thin
layer chromatography) profile. Likewise, there are differences in the visible
absorption spectra of the methanol extracts of the three plant species (Fig. 3).

Spectrum Abs

000

Oe camiaicallus
\ carrot }

400 700

Figure 3. Absorption spectra of the methanol extracts of camia callus, squash and carrot.

Variations in Tissue Development and Product Elaboration of Hedychium coronarium 45

The production of the pigment in camia callus might be due to
the composition of the macroelements in the medium as this was the only
difference between the media. [Succeeding camia floral tube cultures on
mWB medium initiated and maintained in growth room of 16-hr light at
16.0 umolS"'m” provided by fluorescent lamps and in controlled temperature
range of 17-24 °C also produced orange pigmented callus.] The possibility
of osmotic value difference, however, was not discounted. An experiment
using mannitol as osmolyticum will be pursued to check this factor.

The pigment in camia callus grown on MWB medium is likely mainly
carotenoids, but this is yet to be ascertained. Lately, the researcher was
able to secure a flowering and fruiting specimen of H. philippinense. The
flowers of this epiphyte are yellow and the fruits are orange reaching 6 cm
at maturity. In the absence of H. coronarium fruit, pigment of this species’
fruit will be used as reference in the further analysis of the camia callus
pigment. Modifications in the TLC procedure and in the protocol to obtain
absorption spectra of the pigment will be pursued.

Acknowledgements

The author would like to thank the organizers, Singapore Botanic Gardens,
of the 4" International Symposium on the Family Zingiberaceae for enabling
her to participate in the symposium and PCARRD-DOST (Philippine
Council for Agricultural Research and Development — Department of
Science and Technology) for partial funding support of this work.

References

Braun, A.C. and H.N. Wood. 1962. On the activation of certain essential
biosynthetic systems in cells of Vinca rosea L. Proceedings of the
National Academy of Sciences (Washington) 48:1776.

Cardenas, L.B. 1983. Alkaloid production in intact parts and floral tissue
culture of Catharanthus roseus (L.) Don. Kalikasan, Philippine
Journal of Biology 12: 375-384.

Cardenas, L.B. 1993. Somatic Embryogenesis and the Production of Es-
sential Oil Components in some Apiaceae species. Dr. rer. nat.
Thesis, Germany (unpublished).

46 Gard. Bull. Singapore 59 (1&2) 2007

Murashige, T. and F. Skoog. 1962. A revised medium for the rapid growth
and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:

473-497.

Rodriguez-Amaya, D.B. 1999. A Guide to Carotenoid Analysis in Foods.
ILSI Press, USA. 64 pp.

Wagner, H. and S. Bladt. 1996. Plant Drug Analysis. A Thin Layer
Chromatography Atlas. Springer Verlag, Germany. 384 pp.

Gardens’ Bulletin Singapore 59 (1&2): 47-56. 2007 AT

Total Phenolic Content and Antioxidant Activity
of Leaves and Rhizomes of Some Ginger Species in
Peninsular Malaysia

E.W.C. CHAN, Y.Y. LIM AND T.Y. LIM

School of Arts and Sciences, Monash University Sunway Campus,
Bandar Sunway, 46150 Petaling Jaya, Selangor, Malaysia

Abstract

The total phenolic content (TPC) and antioxidant activity (AOA) of leaves
and rhizomes of five wild and six cultivated ginger species belonging to
seven genera were compared. Altitudinal variation in leaf TPC and AOA of
four species of Etlingera Giseke was also studied. TPC was measured using
the Folin-Ciocalteu method. AOA was measured using the1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging assay and expressed as ascorbic
acid equivalent antioxidant capacity (AEAC). Of the 11 wild and cultivated
species screened, leaves of Etlingera had the highest TPC and AEAC, which
were seven to eight times higher than those of rhizomes. Eight species
had significantly higher leaf TPC and/or AEAC than rhizomes. Leaves of
highland populations of Etlingera species had higher values than those of
lowland counterparts.

Introduction

Rhizomes of gingers (Zingiberaceae) are widely consumed as spice or
condiments (Larsen et al., 1999; Sirirugsa, 1999). Major commercially
cultivated species in Peninsular Malaysia are Zingiber officinale Roscoe,
Curcuma longa L. and Alpinia galanga (L.) Willd. As traditional medicine,
rhizomes are consumed by women during ailment, illness and confinement
(Larsen et al., 1999; Ibrahim et al., 2006). They are also taken as carminative
for relieving flatulence.

Leaves of gingers have also been used for food flavouring. In
Peninsular Malaysia, leaves of Curcuma longa are used to wrap fish before
steaming or baking (Larsen et al., 1999). The leaves of Kaempferia galanga
L. and Curcuma longa are ingredients of spicy fish and meat dishes. Some
tribal natives use leaves of Elettariopsis slahmong C.K. Lim to flavour cuisine
of wild meat and fish (Lim, 2003). In Okinawa, Japan, leaves of Alpinia

48 Gard. Bull. Singapore 59 (1&2) 2007

zerumbet (Pers.) B.L. Burtt & R.M. Sm. are traditionally used to wrap rice
cakes and are commercially sold as herbal tea.

Past studies on the antioxidant activity of wild and cultivated
ginger species were confined to rhizomes (Jitoe et al., 1992; Habsah et al.,
2000; Zaeoung et al., 2005). Although their leaves have been used for food
flavouring, hardly any research has been done on their antioxidant activity.

Antioxidants are molecules that are able to scavenge free radicals
or prevent their generation. Phenolic compounds, in general, are able to
scavenge free radicals or chelate metal ions to prevent generation of free
radicals. Free radicals have been implicated in the pathogenesis of more
than 50 diseases (Percival, 1996). Currently, there is much interest in herbs
and spices as sources of antioxidants.

In our present study, the total phenolic content (TPC) and antioxidant
activity (AOA) of leaves and rhizomes of five wild and six cultivated ginger
species were compared. Altitudinal variation in leaf TPC and AOA of
species of Etlingera was also studied.

Materials and Methods

Species studied

Five wild and six cultivated ginger species were screened for TPC and AOA.
Wild species studied were Etlingera maingayi (Baker) R.M. Smith, Alpinia
malaccensis vat. nobilis (Ridl.) I.M. Turner, Elettariopsis slahmong C.K. Lim,
Zingiber spectabile Griff. and Scaphochlamys kunstleri (Baker) Holttum.
Cultivated species studied were Etlingera elatior (Jack) R.M. Smith, Alpinia
galanga (L.) Willd., Zingiber officinale Roscoe, Curcuma longa L., Curcuma
zanthorrhiza Roxb. and Boesenbergia rotunda (L.) Mansf. For each species,
leaves and rhizomes of three plants were sampled.

For wild species, leaves and rhizomes of Alpinia malaccensis var.
nobilis, Zingiber spectabile and Scaphochlamys kunstleri were sampled from
plants growing at Forest Research Institute Malaysia (FRIM) in Selangor,
those of Elettariopsis slahmong from Bukit Lagong in Selangor, and those
of Etlingera maingayi from Janda Baik in Pahang. Voucher specimens of
wild species studied were deposited at the FRIM herbarium (KEP).

For cultivated species, leaves and rhizomes of Etlingera elatior and
Curcuma longa were sampled from plants found at FRIM, those of Alpinia
galanga and Zingiber officinale from Bukit Maluri in Kepong, and those of
Curcuma zanthorrhiza from Damansara Utama in Petaling Jaya. Plants of
Boesenbergia rotunda were purchased from a nursery in Sungai Buluh in
Selangor. Rhizomes of Alpinia galanga, Zingiber officinale and Curcuma
longa purchased from the supermarket were also screened. Voucher

Phenolic Content and Antioxidant Activity of Leaves and Rhizomes of Ginger 49

specimens of cultivated species studied were deposited at KEP.

TPC and AOA of leaves of lowland and highland populations of
four Etlingera species were compared. The species studied were Etlingera
elatior (Jack) R.M. Sm., Etlingera fulgens (Ridl.) C.K. Lim, Etlingera
littoralis (J. KOnig) Giseke and Etlingera rubrostriata (Holttum) C.K. Lim.
Their identification was based on taxonomic descriptions and photographic
illustrations of Lim (2000 & 2001) and Khaw (2001). Leaves of highland
populations were sampled from Janda Baik and Genting Highlands
in Pahang and from Ulu Gombak in Selangor, while those of lowland
populations were sampled from FRIM. For each location, mature leaves
were sampled from three different plants per species. Voucher specimens
of Etlingera species studied were deposited at KEP. Altitude of locations,
where the populations were sampled, was measured using a Casio altimeter
(Model PRG-70-1VDR).

Extraction of samples

Fresh leaves and rhizomes (1 g) were powdered with liquid nitrogen in a
mortar and extracted by methanol (50 ml), with continuous swirling for one
hour at room temperature. Extracts were filtered and stored at -20°C for
further use. Analysis of methanol extracts was done in triplicate for each
species.

Total phenolic content

Total phenolic content (TPC) was measured using the Folin-Ciocalteu
method (Kahkonen et al., 1999). Samples (300 ul in triplicate) were
introduced into test tubes followed by 1.5 ml of Folin-Ciocalteu’s reagent
(10 times dilution) and 1.2 ml of sodium carbonate (7.5% w/v). The tubes
were allowed to stand for 30 min before absorption at 765 nm was measured.
Total phenolic content was expressed as gallic acid equivalent (GAE) in
mg/100 ¢g material. The calibration equation for gallic acid was y = 0.0111x
— 0.0148 (R = 0.9998).

Antioxidant activity

Antioxidant activity (AOA) was measured using the 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging assay used by Leong and Shui
(2002) and Miliauskas et al. (2004) with slight modification. Defined amounts
of the extract were added to 3 ml of DPPH (3.9 mg/100 ml methanol). The
DPPH solution was then allowed to stand for 30 min before absorbance
was measured at 517 nm. All spectrophotometric measurements were made
with methanol as blank. An appropriate dilution of the DPPH solution
was used as negative control, 1.e., methanol in place of the sample. Results

50 Gard. Bull. Singapore 59 (1&2) 2007

were expressed as ascorbic acid equivalent antioxidant capacity (AEAC)
in mg/100 g calculated from the ICso (inhibitory concentration in mg/ml of
plant material necessary to reduce the absorbance of DPPH by 50%) using
the following formula:

AEAC (mg AA/100 g) = IC socascorbatey/ 1Cso(cample) Xx 100,000

The ICso of ascorbate used for calculation of AEAC was 0.00387 mg/ml.

Results and Discussion

Leaves and rhizomes of wild and cultivated species

Results from screening of five wild species showed that leaves of Etlingera
maingayi had significantly higher TPC and AEAC than those of Alpinia
malaccensis var. nobilis, Elettariopsis slahmong, Zingiber spectabile and
Scaphochlamys kunstleri (Table 1). Rhizomes of Alpinia malaccensis var.
nobilis had the highest values. Leaves of Elettariopsis slahmong, Etlingera
maingayi and Scaphochlamys kunstleri showed significantly higher values at
P < 0.05 than rhizomes. Leaves of other wild species were only marginally
higher than rhizomes.

For six cultivated species screened, leaf and rhizome TPC and AEAC
were highest in Etlingera elatior and Curcuma longa, respectively (Table 2).
In five species, leaves had significantly higher TPC and/or AEAC at P < 0.05
than those of rhizomes. Exceptions were AEAC of Alpinia galanga, and
TPC and AEAC of Curcuma longa where rhizomes showed higher values
than leaves. The values of Curcuma longa were highly variable between
rhizomes. For Alpinia galanga, Curcuma longa and Zingiber officinale,
differences existed between collected rhizomes and those purchased from
the supermarket. This implies that there is variability in TPC and AEAC
between different cultivars.

In general, leaves of wild and cultivated Etlingera species contain
the most antioxidants by having the highest TPC and AEAC. Values were
1110 mg GAE/100 g and 963 mg AA/100 g for Etlingera maingayi (Table 1),
and 2390 mg GAE/100 g and 2280 mg AA/100 g for Etlingera elatior (Table
2) respectively. The outstanding leaf TPC and AEAC of both Etlingera
maingayi and Etlingera elatior were seven to eight times higher than those
of rhizomes.

There are very few studies comparing between the AOA of leaves
and rhizomes of ginger species. Agnaniet et al. (2004) reported that essential
oils extracted from leaves of Aframomum giganteum K. Schum. had higher
AOA than rhizomes. Contrary to our results, Katsube et al. (2004) reported
higher TPC and AOA in rhizomes of Zingiber officinale than leaves. It is

Phenolic Content and Antioxidant Activity of Leaves and Rhizomes of Ginger By |

Table 1. Total phenolic content (TPC) and ascorbic acid equivalent antioxidant capacity
(AEAC) of leaves and rhizomes of five wild ginger species.

Species and location Voucher Plantpart TPC AEAC
number (mg GAE/100¢g) (mg AA/100 g)

Alpinia malaccensis var. ECO! Leaves 744 + 61° 800 + 62 *
nobilis - FRIM Rhizomes 564+ 209° 745 + 342 *
Elettariopsis slahmong - EC02 Leaves 346 + 45° 209-5167
Bukit Lagong Rhizomes 219+57° 197 + 76°
Etlingera maingayi - EC06 Leaves TnOle 93° 963 + 169 *
Janda Baik Rhizomes 160+52° 122+53°
Scaphochlamys kunstleri- |ECO08 Leaves 203721 * bs ees hee
FRIM Rhizomes 73+3° 14+2°
Zingiber spectabile - BCU Leaves 242 121 + 24°
FRIM Rhizomes 157+ 100° 124 + 109°

Values of TPC and AEAC are means + SD (n = 3). For column of each species, values
followed by the same letter (a-b) are not significantly different at P < 0.05 measured by the
Tukey HSD test. ANOVA does not apply between species.

not known whether their comparisons were based on samples from same or
different plants.

This is probably the first study where TPC and AOA of leaves and
rhizomes of gingers have been systematically compared. For most of the
species screened, TPC and/or AEAC of leaves were significantly higher
than rhizomes.

Antioxidants are secondary metabolites, which form part of the
plant’s protective mechanism against free radicals. In Zingiberaceae, it is
generally believed that antioxidants and other secondary metabolites are
transported to the rhizomes where they are accumulated. This implies that
rhizomes would have higher AOA than other plant parts. However, results
of this study showed that this might not be the case.

Photosynthesis and respiration are physiological processes
comprising several free radical intermediates. Exposure to sunlight can
also increase the amount of free radicals. Leaves therefore require much
more free radical scavengers than other plant parts. Similarly, Frankel and
Berenbaum (1999) found that foliage of tropical forest plants produced more
antioxidants when exposed to elevated light conditions. This observation
may also apply to species of Etlingera, which have the highest leaf TPC and
AEAC. Etlingera plants grow in gaps of disturbed forest and are continually

Gard. Bull. Singapore 59 (1&2) 2007

Table 2. Total phenolic content (TPC) and ascorbic acid equivalent antioxidant capacity
(AEAC) of leaves and rhizomes of six cultivated ginger species.

Species and location Voucher Plant part EPC AEAC
number (mg GAE/100g) (mg AA/100 g)
Alpinia galanga - EC10 Leaves 366 + ey 72240
Bukit Maluri Rhizomes 10222 96 +6
Supermarket Rhizomes 214 + 20 168 + 13
Boesenbergia rotunda- EC11 Leaves 260+8° Lod 3 2,
Sungai Buluh Rhizomes 197 +50" 89 +7
Curcuma longa - EC12 Leaves 230+ 19° , 113 +18° y
FRIM Rhizomes 534 + 205 390 + 127
Supermarket Rhizomes 386 + 219 219,183
Curcuma zanthorrhiza- EC13 Leaves 503-57 : 26) +39 7
Damansara Utama Rhizomes 250+ 92 134 +21
Etlingera elatior - EC14 Leaves 2390 + 329 ; 2280 + 778°
FRIM Rhizomes 326 + 76 295 +96
Zingiber officinale - EC15 Leaves 291 + 18 ; 96+7°
Bukit Maluri Rhizomes 157 +18 84+3°
Supermarket Rhizomes 184+ 11 167 29

exposed to direct sunlight (Poulsen, 2006). Furthermore, leaves of Etlingera
are long lasting and do not abort. This may be due to an efficient protective
mechanism delaying senescence in leaves, which is partly attributed to
oxidative stress.

Altitudinal variation in leaves of Etlingera species

Leaves of all four species of Etlingera sampled from highland populations
were found to have higher TPC and AEAC than lowland counterparts (Table
3). Leaves of Etlingera rubrostriata, Etlingera elatior and Etlingera fulgens
showed significantly higher values at P < 0.05, while Etlingera littoralis was
marginally higher. Highest TPC and AEAC were found in the leaves of
highland populations of Etlingera elatior with values of 3550 mg GAE/100 g
and 3750 mg AA/100 g, and of Etlingera rubrostriata with values of 3480 mg
GAE/100 g and 3540 mg AA/100 g, respectively.

Phenolic Content and Antioxidant Activity of Leaves and Rhizomes of Ginger 55

Table 3. Total phenolic content (TPC) and ascorbic acid equivalent antioxidant capacity
(AEAC) of leaves of four Etlingera species sampled from highland and lowland locations.

Species and location Voucher Altitude Moisture sue AEAC
number (masl) content(%) (mg GAE/100g) (mg AA/100 g)
Ealingera elaiior - EC03 400 66.1+2.0 3550 +304" S150 = 505"
Janda Baik 100 2390 + 329 2280 + 778
FRIM
eee ES eet i400 743401 2270431", 2030 + 126°
Janda Baik 100 1280 + 143 845 + 158
FRIM
Ze EG0sle 1800 WM2e08" (2810 243° 2930 + 220°
oo ee ads 100 2340 + 386° 2220 + 913°
FRIM
Etlingera rubrostriata- C7 300 71.6 + 2.8 3480 + 390 3540 + 401°
Ulu Gombak 100 2430 + 316 2640 + 508°
FRIM

Values of TPC and AEAC are means + SD (n = 3). For columns of each species, values
followed by the same letter (a-b) are not significantly different at P < 0.05 measured by the
Tukey HSD test. ANOVA does not apply between species.

Higher altitudes seem to trigger an adaptive response in the species
of Etlingera. The higher leaf TPC and AEAC of highland populations over
lowland counterparts might be due to environmental factors such as higher
UV-B radiation and lower air temperature. Plants protect themselves
from oxidative damage due to UV exposure by producing antioxidative
compounds (Larson, 1988). Enhanced UV-B radiation induces greater
production of phenolic compounds (Bassman, 2004). Enzymes associated
with the synthesis of phenolics are produced in greater quantities or show
increased activity (Chalker-Scott & Scott, 2004). Phenylalanine ammonia
lyase (PAL) of the phenylpropanoid pathway is up-regulated resulting in
the accumulation of flavonoids and anthocyanins, which have free radical
scavenging ability (Jansen et al., 1998). Low temperatures have also been
shown to enhance PAL synthesis and activity in a variety of plants, leading
to an increase in flavonoids and other phenolics (Chalker-Scott & Scott,
2004).

Conclusion

Based on five wild and six cultivated ginger species belonging to seven
genera, highest TPC and AOA were found in the leaves of Etlingera. For

54 Gard. Bull. Singapore 59 (1&2) 2007

most species screened, leaf TPC and/or AEAC were significantly higher than
those of rhizomes. Rhizomes from different cultivars showed variability in
TPC and AEAC. Leaves of highland populations of Etlingera had higher
values than lowland counterparts. Of the four species studied, highest TPC
and AEAC were found in the leaves of highland populations of Etlingera
elatior and Etlingera rubrostriata.

Acknowledgements

The authors are thankful to botanists in FRIM, Saw, L.G. and Lau, K.H.,
who assisted in the identification of wild ginger species. Chung, R.C.K. and
Sam, Y.Y. facilitated the deposition of voucher specimens in the FRIM
herbarium. The financial support by Monash University Sunway Campus is
gratefully acknowledged.

References

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Gardens’ Bulletin Singapore 59 (1 &2): 57-64. 2007 57

Studies on the Rhizome Oils from Four Hedychium
Species of South India: A Chemotaxonomic Approach

M. DAN ',B. SABULAL |, V.GEORGE ' AND P PUSHPANGADAN —

‘Tropical Botanic Garden and Research Institute,
Pacha-Palode, Thiruvananthapuram - 695 562, Kerala, India
“Amity Institute for Herbal and Biotech Product Development,
Noida, New Delhi, India

Abstract

The genus Hedychium J. Konig (Zingiberaceae) with about 80 species has
only four members in south India, viz., H. coronarium J. Konig, H. flavescens
Carey ex Roscoe, H. spicatum var. acuminatum (Roscoe) Wall., and the
endemic, H. venustum Wight. The chemical compositions of essential oils
from the rhizomes of these four Hedychium species and their morphologies
have been compared. The rhizome oils were characterized by analytical gas
chromatography and gas chromatography-mass spectroscopy. The number
of identified constituents in the rhizome oils of H. coronarium, H. flavescens,
H. spicatum var. acuminatum and H. venustum were 24 (99.7%), 27 (98.8% ),
41 (98.9%) and 57 (99.1%) respectively. 1,8-cineole, B-pinene and linalool
constituted 70-75% of these rhizome oils. 1,8-cineole was the single major
constituent in the rhizome oils of H. coronarium (48.7%), H. venustum
(45.4%), and H. spicatum var. acuminatum (44.3% ). B-pinene (43.6%) was
the major component in the rhizome oil of H. flavescens. The percentages
of sesquiterpenes in these oils were H. venustum (24.0%), H. spicatum var.
acuminatum (22.1%), H. coronarium (3.1%) and H. flavescens (1.3%). Oil
yields from the rhizomes of H. venustum, H. spicatum var. acuminatum and
H. coronarium were comparable (0.13-0.16%), but that from the rhizomes
of H. flavescens was substantially low (0.05%). H. venustum and H. spicatum
var. acuminatum are morphologically similar and significantly different from
H. flavescens. The chemical data on essential oils are in good agreement
with the relative morphological features of these four Hedychium species
and thus chemotaxonomically significant.

Introduction

The genus Hedychium J. K6nig (Zingiberaceae) represents the spectacular

58 Gard. Bull. Singapore 59 (1&2) 2007

‘Ginger lilies’ comprising over 80 species distributed in India, China and
South East Asia. In India it has about 50 species with about 17 endemics
(Sabu, 2000). The genus is represented in south India by only four species,
viz., H. coronarium J. Konig, H. flavescens Carey ex Roscoe, H. spicatum var.
acuminatum (Roscoe) Wall. and H. venustum Wight, of which H. venustum
is endemic to this region. H. coronarium is widely cultivated as a garden
plant and the rest are restricted to hilly slopes at altitudes ranging from 1000
to 1800 m. All the species have fleshy, branched rhizomes characteristically
aromatic due to the presence of essential oils. Essential oils are complex,
volatile, aromatic, heterogeneous mixtures containing mostly mono- and
sesquiterpenes and their derivatives. Inter-relationships of these four
South Indian taxa of Hedychium were established by correlating their
morphological characters and the percentages and distribution of mono-
and sesquiterpenes in their rhizome oils.

Materials and Methods

Flowering specimens and rhizomes of Hedychium flavescens, H. spicatum vat.
acuminatum and H. venustum were collected from established populations
in natural habitats at Devikolam, Munnar (Idukki District) and Ponmudi
(Thiruvananthapuram District), Kerala State in South India. H. coronarium
was collected from an established population in Tropical Botanic Garden
and Research Institute (TBGRI), Thiruvananthapuram. All specimens
were collected in September of 2004, and their vouchers (Mathew 54620,
54609, 54622 and 54624, respectively) were deposited in the Herbarium of
TBGRI (TBGT). Seventeen morphological characters from each species
were compared (Table 1), of which quantitative characters are the mean
values of six measurements taken from different shoots within a clump. The
measurements of leaves (length, breadth and area) were determined from
middle leaves of different shoots.

Essential oils of these four taxa were isolated from fresh rhizomes
by hydrodistillation in a Clevenger-type apparatus for 3 hrs. Chemical
constituents of these oils were analysed by analytical gas chromatography
(GC-FID) and gas chromatography-mass spectroscopy (GC-MS) as
described in Sabulal et al (2007). GC-FID of rhizome oils were carried out on
a Nucon 5765 gas chromatograph with a SE-30 10% Chromosorb-W packed
stainless steel column (2 m x 2 mm). Oven programme consisted of 60°C
(S min), 60°-260°C (5°C/min), 260°C (10 min); carrier gas — nitrogen, flow
rate 40 ml/min; injector temperature 240°C; detector temperature 240°C.
GC-MS analysis of these oils were performed by splitless injection of 1.0
ul of each oil on a Helwett Packard 6890 gas chromatograph fitted with a

Rhizome Oils from Four Hedychium Species of South India

Ly,

Table 1. Comparison of morphological characters of four species of Hedychium. See text for

the species names.

Characters

Shoot length (cm)
Leaf length (cm)
Leaf breadth (cm)
Leaf area (cm’)*

Ligule length (cm)*
Petiole length (cm)*
Spike length (cm)*
Spike nature
Peduncle length (cm)*
Bract length (cm)*
Bract breadth (cm)*
Corolla lobe colour
Staminode colour
Lip length (cm)*
Lip shape

Lip colour

Stamen colour

H. cor.

120 - 180
40-50
8-12
206
She
BO
10.3
Dense
| es
6.0
ea!
White
White
6.0
Obovate
White
White

*Values are mean of six measurements

H. fla.

200- 260
35-45
10-14

258
30)
eis,
15.6
Dense
ey
5.0
pa,
Yellow
Yellow
5.0
Obovate
Yellow
Yellow

H, spi. ac.

100-140
24-30
6-8
187
0.94
1%
19
Lax
2.0
23
1.0
Yellow
White
pi
Oblanceolate

White
Red

H. ven.

100- 180
35-40
10-12

pAlN
1.6
a5
23
Lax
od,

j dep)
1.0
Yellow
White
De
Oblanceolate
White
Red

cross-linked 5% PH ME siloxane HP-5 MS capillary column, 30 m x 0.32
mm, 0.25 ut coating thickness, coupled with a model 5973 mass detector.
GC-MS operation conditions: injector temperature - 220°C; transfer line -
240°C; oven temperature programme - 60° to 243°C (3°C/min); carrier gas
-He at 1.4 ml/min. Mass spectra: Electron Impact (EI) mode 70 eV, ion
source temperature 240°C. Individual components were identified by Wiley
275.L database matching, comparison of mass spectra with published data
and by comparison of their relative retention times. Morphological inter-
relationship of the four species was established by calculating the Simple
Similarity Coefficient (Sr.) (Table 2) using the following formula proposed
by Sokal & Sneath (1963). The Sr. of chemical interrelationship was also
calculated using the same formula (Table 3).

common characters of two species

Sr= x100
common characters characters characters
of two species + specific to one + specific to other
species species

60 Gard. Bull. Singapore 59 (1&2) 2007

Results and Discussion

Morphological interrelationship based on Simple Similarity Coefficient of
these four Hedychium species showed a maximum relationship between
H. spicatum var. acuminatum and H. venustum with Sr. 36% (Table 2). The
chemical inter-relationships between these four taxa were also determined
based on the constituents identified from their rhizome oils by GC-MS
analysis (Sabulal et al, 2007) (Table 3). H. flavescens is morphologically and
chemically distinct from all others and showed maximum relationship to H.
coronarium with Sr. 21% and 50% (Tables 2 & 3). The percentage of oil
yields was calculated based on fresh weight of rhizomes. The oil yield was
highest in H. venustum (0.16%) and lowest in H. flavescens (0.05%). The oil
yields in H. spicatum var. acuminatum and H. coronarium were 0.13% and
0.13%, respectively.

Table 2. Simple Similarity Coefficient on morphological data between four species of Hedy-
chium in South India. See text for the species names.

A cons fie, va sp aes Gea.

100

Table 3. Simple Similarity Coefficient on chemical data between four species of Hedychium
in South India. See text for the species names.

H.. cor, A. fla. 4 dd SPL GAC te:
100 50 a3 23

28 24
66

Rhizome Oils from Four Hedychium Species of South India 61

Fifty-seven constituents (99.1%) were identified in the rhizome
oil of H. venustum, forty-one constituents (99.2%) from H. spicatum var.
acuminatum, twenty- five constituents (98.2%) from H. flavescens, and
twenty-three constituents (97.5% ) from H. coronarium. Thirteen constituents
were found common in all the four species. The major constituents in
these species were the monoterpenes 1,8-cineole, B-pinene and linalool,
constituting 70-75 % of these oils (Sabulal et al,2007). 1,8-Cineole is the major
constituent in three species, while B-pinene is the major one in H. flavescens
(Fig. 1) showing the divergence of monoterpene metabolic pathways in H.
flavescens in agreement with its morphological and chemical dissimilarity
with others. Out of the twenty-five constituents identified from H. flavescens,
sixteen are common to H. coronarium and thus, the Sr. on chemical data
between the two is 50% (Table 3). Chemical relation of H. flavescens with
H. spicatum var. acuminatum 1s 28% because, constituents found common
to them are fifteen out of the total forty-one constituents identified from the
latter and with H. venustum, it is only 24% (Table 3) as sixteen constituents
are common out of the total fifty-seven. Lower percentage of B-pinene
and higher percentage of 1,8-cineole in H. spicatum var. acuminatum and
H. venustum (Fig. 1) support their close similarity in morphological and
chemical inter-relationships. The monoterpenes, viz., myrcene, limonene,
p-cymene, camphene and jy-terpinene, were identified from all the four
taxa studied here (Fig. 2). These monoterpenes were reported as common
constituents in all Hedychium species previously studied (Medeiros et al.,
2003). The relative percentages of these monoterpenes are highest in H.
flavescens (Fig. 2).

@ B-pinene
B 1,8-cineole

0 linalool

H.coro. H.flav. H.spic. H.venu.

Figure 1. Percentage of major monoterpenes found in four species of Hedychium in South
India.

62 Gard. Bull. Singapore 59 (1&2) 2007

Z -
g -
5 & camphene

| & myrcene
4 | @ p-cymene
3 O limonene
na @ y-terpinene
1-

H.coro. H. flav. H.spic. H.venu.

Figure 2. Percentage of minor monoterpenes common to four Hedychium species in South
India.

Sesquiterpenes, with farnesyl pyrophosphate (FPP) as its precursor, are
more evolved in the biosynthetic pathway than monoterpenes, with geranyl
pyrophosphate (GPP), asits precursor. The sesquiterpene distribution is more
dominant in H. venustum compared to others. The number and percentage
of sesquiterpenes and their derivatives in H. venustum were 38 and 24.0%,
respectively. The number and percentages of sesquiterpenes in H. spicatum
var. acuminatum, H. coronarium and H. flavescens were 23 (22.1%), 4 (3.1%)
and 6 (1.3%) respectively (Fig. 3). The role of essential oil constitunets as
markers in chemotaxonomic studies is described by Hegnauer (1982) and
very well proved by many botanists as well as chemists. Among these four
south Indian taxa of Hedychium, H. venustum closely resembles H. spicatum
var. acuminatum morphologically as well as in mono- and sesquiterpene
distribution indicating their phylogenetic affinity. Thirty-nine constituents
are common in the essential oils of these two species and resulted in 66%
chemical similarity between them (Table 3). The current data also suggest
that transformation of the farnesyl pyrophosphate to various sesquiterpenes
by terpenoid synthases is most advanced in H. venustum, followed by H.
spicatum var. acuminatum. The major monoterpene component, B-pinene,
and relatively very low numbers of sesquiterpenes in the oil suggest the
sharp difference in mono- and sesquiterpene pathways in H. flavescens and
also indicate its primitiveness in phylogeny.

Rhizome Oils from Four Hedychium Species of South India 63

i No. of sesquiterpenes
m % of sesquiterpenes

H. coro. H. flav. H. spic. _—_H. venu.

Figure 3. Number and percentage of sesquiterpenes found in four species of Hedychium in
South India.

Conclusion

The major constituents of the rhizome oil of all the four south Indian
Hedychium species are monoterpenes, viz., 1,8- cineole, B-pinene and linalool
(70-75% ). The inter-relationship of the four taxa based on chemical data is
well in tune with that of morphological data. H. venustum is morphologically
closer to H. spicatum var. acuminatum and they also show 66% chemical
similarity to each other. H. flavescens is distinct from others morphologically
as well as chemically. The species-specific chemical profiles of essential oils
from these four Hedychium taxa are chemotaxonomically significant.

References

Hegnauer, R. 1982. Distribution and chemotaxonomy of essential oils, pp.
1-23. In: Atal, C. K. and Kapur, B. M. (eds.) Cultivation and Utilization of
Aromatic Plants. RRL, CSIR, Jammu-Tawi.

Medeiros, J. R., L.B. Campos, S.C. Mendonca, L.B. Davin and N.G. Lewis.
2003. Composition and antimicrobial activity of the essential oils from
invasive species of the Azores, Hedychium gardnerianum and Pittosporum
undulatum. Phytochemistry 64: 561-565.

Sabu, M. 2000. Hedychium spicatum Ham. ex Smith var. acuminatum
(Roscoe) Wall. — a new record for peninsular India. Rheedea 10: 73-76.

64 Gard. Bull. Singapore 59 (1&2) 2007

Sabulal, B., V. George, M. Dan and N.S. Pradeep. 2007. Chemical composition
and antimicrobial activities of the essential oils from the rhizomes of four
Hedychium species from south India. Journal of Essential Oil Research

19: 93-97.

Sokal, R. R. and P.H.A. Sneath. 1963. Principles of Numerical Taxonomy.
Freeman, San Fransisco & London.

Gardens’ Bulletin Singapore 59 (1&2): 65-70. 2007 65

Micropropagation of Boesenbergia pulchella (Ridl.)
Merr., a Potential Ornamental Plant

M.N. HAMIRAH |',H.B.SANI', P.C. BOYCE’ AND S.L. SIM '!

‘Institute of Biodiversity and Environmental Conservation, University of
Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
* Malesiana Tropicals, Suite, 9-04,Tun Jugah Tower, No 18 Jin Tunku Abd Rahman,
90100 Kuching, Sarawak, Malaysia

Abstract

Shoot tips of Boesenbergia pulchella (Ridl.) Merr. were cultured on Gamborg
B5 medium containing 30% (w/v) sucrose and 2.8% (w/v) Gelrite. Various
concentration of plant growth regulators (PGR) were supplemented to B5
media, i.e., 6-benzylaminopurine (BAP) at 1-4 mg/l alone or in combination
with o-naphtalene acetic acid (NAA) at 0.1 mg/l or thidiazuron (TDZ) at 0.1-
0.7 mg/l. Multiple shoot formation were found on both media supplemented
with TDZ and BAP. A maximum of 11 shoots were produced after treatment
with TDZ at 0.3 mg/l, which were the highest among other treatments.
Acclimatization were conducted on (1:1 v:v) soil and vermiculite.

Introduction

Boesenbergia pulchella (Ridl.) Merr. is a small herb known as ‘jerangau’
locally. This plant is a member of one of the most advanced monocotyledonous
plant family, Zingiberaceae. Commonly, this attractive plant can be
found on the forest floor. They have beautiful glossy green leaves and the
inflorescences are lanceolate on separate leafless shoot. The flower is small,
open from apex to bottom with white and red labellum. Since they possess
beautiful flower and foliage, they can be introduced as ornamental plants
and have the potential to be commercialized, either as indoor potted plant
or garden plant, even for landscaping purposes.

To achieve the goal of commercializing this plant, a large number of
planting materials are needed. Propagation through conventional technique
via rhizome cutting is very slow. This plant will produce shoot once they
flower, which takes about three to four months after the shoot emerges.
The highest number of shoot produced is two per mother plant, usually it is
just one, and this restricts the use of conventional means. Micropropagation

66 Gard. Bull. Singapore 59 (1&2) 2007

enables mass production of clone and thus could satisfy the demand for
planting material. The other benefit of using this technique is that the
plantlet is disease-free and true-to-type.

Successful micropropagation of anumber of species in Zingiberaceae
such as Zingiber officinale Roscoe, Curcuma longa L., Kaempferia galangal
L. and Alpinia galanga (L.) Willd. were reported (Bhagyalakshmi and
Singh, 1988; Shirgukar ef al., 2001; Shirin et al., 2000; Borthakur et al.,
1999). However, to date, no successful micropropagation protocol has been
reported for this species.

The present investigation is an attempt towards establishing a
reliable in vitro regeneration protocol for Boesenbergia pulchella Ridley
for use in large-scale propagation. Different concentrations of BAP with or
without NAA and TDZ were tested to find the best PGR that can induce
the optimum multiplication rate for this species.

Materials and Methods

Explants sources and sterilization

The stock plants for this study were collected from Gunung Ampungan in
Kota Samarahan district. Rhizome buds between 1-1.5 cm were selected as
initial explants. The fresh buds collected were cleaned of soil dirt and left
under running tap water for one to one and a half hour. Then the buds were
immersed in 75% (w/v) ethanol for one minute. Without rinsing, they were
agitated in 30% (w/v) Clorox (5.25% w/v sodium hypoclorite) added with
0.1ml/l Tween 20 for 20 minutes with constant agitation. After that they were
rinsed with sterile-distilled water four times. Under aseptic condition the
buds scale were peeled-off and they were trimmed to about 0.5 cm long.

Establishment of axenic culture

The trimmed buds were cultured on Gamborg B5 medium, gelled with 2.8¢/1
Gelrite, 30% sucrose. The pH was adjusted to 5.7-5.8 with IN KOH or 0.1 N
HCL prior to autoclaving. Tetracycline at 10 mg/l and 1 ml/I Plant Preserva-
tive Mixture (PPM) were added to the medium to reduce contamination.
After 15 days, the axenic culture were cut into half and subcultured onto
BS medium supplemented with BAP at | mg/l for 4 weeks to induce more
shoots. The shoots were subcultured on B5 media for 2 weeks before they
were used in subsequent experiment.

To study the effects of different types and concentrations of PGR on
shoot multiplication, different treatments were used, i.e., BAP at 1, 2,3 and
4 mg/l alone or each added with 0.1 mg/l NAA and TDZ at 0.1, 0.3, 0.5 and
0.7 mg/l.

Micropropagation of Boesenbergia pulchella 67

Rooting and acclimatization

Rooting was induced on B5 medium without plant growth regulator. Plantlet
at about 6-8 cm height were taken out from the vessel and washed thoroughly
with tap water before they were transferred to plastic pot containing 1:1 soil
and vermiculite. The plants were covered with plastic to retain moisture.

Results and Discussion

After one week, the explants turned greenish from white. For establishment
of axenic culture, BS medium with and without tetracycline and PPM
were used. However, addition of tetracycline and PPM did not reduce
contamination satisfactorily if compared with explants cultured on B5
medium only. About 50% of the explants were discarded for bacterial
contamination. The result showed that tetracycline was not beneficial to
control bacterial contamination for this species.

Induction of multiple shoots

In this study, excised shoot obtained from in vitro raised plants were used
as explants. Shoots were divided into half prior to culture on different
treatment of PGR. Treatment with BAP alone or in combination with NAA
showed variability in terms of number of shoots produced per explant (Table
1). Optimum concentration for shoot multiplication was found on medium
supplemented with BAP at 3 mg/l, which produced 6.6 shoots per explants.
However, in terms of number of days the first bud sprouted, the duration
was not really different between one treatment to another. After two weeks,
at least one bud sprouted for each treatment. The basal part of the explant
was enlarging before the new buds sprouted from the lateral side.

For treatment with BAP added with NAA 0.1 mg/l, the number of
shoots produced was not much different with the treatment using BAP alone.
The highest number of shoots was obtained on B5 medium supplemented
with BAP at 3 mg/l added with NAA at 0.1 mg/l with a mean of 6.8 shoots
per explant. Hence, this proved that addition of NAA was not beneficial
in increasing the number of shoots. Miachir et al. (2004) also reported a
similar finding on Curcuma zedoaria (Christm.) Roscoe where addition of
NAA with BAP was not effective for shoot multiplication. However, results
from this study showed that explants that were subjected to this treatment
produced roots faster than treatment with BAP alone or TDZ. This was
probably due to the fact that NAA is an auxin that helps in promoting root
development.

In TDZ supplemented media, highest number of shoots obtained
was on medium incorporated with TDZ at 0.3 mg/l. Eleven shoots per

68 Gard. Bull. Singapore 59 (1&2) 2007

explants were developed in the above medium and this was the highest
among other treatments. However, more shoot clusters were formed on
media supplemented with TDZ. These clusters were later subcultured on
fresh medium and were able to regenerate into multiple shoots. The clusters
were first separated into smaller clump, since the regeneration was faster if
compared to larger clump based on the observation.

Table 1. Shoots multiplication under different growth regulators after 12 weeks of culture.

Growth regulator (mg/l) | *No of shoots/explant Days to induction
of new shoots

BAP (1) 3.60 + 0.68
BAP (2) 4.40 + 0.86
BAP (3) 6.60 + 1.03

BAP (4)
BAP (1) + NAA (0.1)

4.20 + 1.20
4.60 + 0.75

BAP (2) + NAA (0.1) 4.00 + 0.84
BAP (3) + NAA (0.1) 6.80 + 1.24
BAP (4) + NAA (0.1) 3.96 + 1.77

TDZ (0.1) 9.20+ 3.44
TDZ (0.3) 11.00 + 1.52
TDZ (0.5) 9.80 + 1.74
TDZ (0.7) 6.00 + 1.14

* Data expressed as mean + SE from 5 replicates

Among the shoot multiplication studies conducted, it is shown
that TDZ was able to regenerate a high number of shoots even at lower
concentration. This accords with Tefera and Wannakrairoj (2000) where
they managed to obtain 15.52 shoots on treatment with TDZ at 0.5 mg/l for
multiplication of Curcuma longa. However for BAP, a higher concentration
is needed to obtain more shoots. In fact, previous studies on other
Zingiberaceae species did use a high BAP concentration. Nayak (2000)
obtained the highest shoot multiplication of Curcuma aromatica Salisb. on
medium supplemented with BAP at 5 mg/l and Samsudeen et al. (2000) used
10 mg/l BAP to induce organogenesis in Zingiber officinale.

Rooting was relatively easy for this species. Vigorous roots were
formed on B5 medium without growth regulator. Plants were successfully
acclimatized with survival rate of 80% after two months.

Micropropagation of Boesenbergia pulchella 69

Figures a-c. Shoots multiplication on BS medium supplemented with BAP at 3 mg/l

after 4,8 and 12 weeks of culture (bar = 0.5, 1 and 2 cm); Figures d-f. Shoots multiplication
on B5 medium supplemented with TDZ at 3 mg/l after 4, 8 and 10 weeks of culture (bar =
i, > and 2.3,cm).

Conclusions

In vitro technique is a useful approach for propagating plants on large scale.
For ginger species, propagation through conventional technique is time
consuming and prone to spread disease by rhizome cuttings.

Shoots multiplication of Boesenbergia pulchella can be obtained
using TDZ and BAP. While TDZ can produce shoots at a lower concentration,
BAP is needed at higher concentration to produce similar results. Addition
of NAA is not beneficial for shoot multiplication of this species.

Acknowledgement

The authors would like to acknowledge the IGS fund (Grant no. IGS (R&D)
16/03) granted by The Ministry of Science, Technology and Innovation
(MOSTI), Malaysia for financial support of the research. We wish to express
our thanks for the collaboration and support of the Sarawak Forestry
Department, notably Datu Cheong Ek Choon, the Sarawak Biodiversity

70 Gard. Bull. Singapore 59 (1&2) 2007

Centre, in particular Datin Eileen Yen Ee Lee and the Forest Research
Centre (Kuching), notably L.C.J. Julaihi Abdullah. The tertiary author wishes
to thank Datuk Amar (Dr) Leonard Linggi Tun Jugah, Graeme Brown and
Dr Timothy Hatch of Malesiana Tropicals Sdn Bhd for their considerable
support and funding of fieldwork in Sarawak

References

BhagyalakskmiandN.S. Singh. 1988. Meristem culture and micropropagation
of a variety of ginger (Zingiber officinale Rosc.) with a high yield of
oleoresin. Journal of Horticultural Science 63: 321-327.

Borthakur, M., J. Hazarika and R.S. Singh. 1999. A_ protocol for
micropropagation of Alpinia galanga. Plant Cell, Tissue and Organ
Culture 55: 231-233.

Miachir, J.I., V.L.M. Romani, A.F. de Campos Amaral, M.O. Mello, O.J.
Crocomo and M. Melo. 2004. Micropropagation and callogenesis of
Curcuma zedoaria Roscoe. Scientific Agriculture 61: 427-432.

Nayak, S. 2000. In vitro multiplication and microrhizome induction in
Curcuma aromatica Salisb. Plant Growth Regulation 32: 41-47.

Samsudeen, K., K. Nirmal Babu, M. Divakaran and P.N. Ravindran. 2000.
Plantregeneration from anther derived callus cultures of gingers (Zingiber
officinale Rosc.) Journal of Horticultural Science and Biotechnology 75:
447-450.

Shirgukar, M.V., C.K. John and R.S. Nagdauda. 2001. Factors affecting in
vitro microrhizome production in turmeric. Plant Cell, Tissue and Organ
Culture 64: 5-11.

Shirin, F.,S. Kumar and Y. Mishra. 2000. In vitro plantlet production system
for Kaempferia galanga, a rare Indian medicinal herb. Plant Cell, Tissue
and Organ Culture 63: 193-197

Tefera, W. and S. Wannakrairoj. 2004. A micropropagation method for
Korarima (Aframomum corrorima (Braun) Jansen). Science Asia 30: 1-
ip

Gardens’ Bulletin Singapore 59 (1&2): 71-88. 2007 vel

Cultivated Gingers of Peninsular Malaysia: Utilization,
Profiles and Micropropagation

H. IBRAHIM ',N. KHALID ' AND K. HUSSIN”

‘Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603
Kuala Lumpur, Malaysia
*School of Environmental and Natural Resource Sciences, Faculty of Science and
Technology, National University of Malaysia, 43600 Bangi, Malaysia

Abstract

There are approximately 160 species of Zingiberaceae belonging to 18
genera in Peninsular Malaysia. Roughly 16-20% are traditionally utilized
by the indigenous folks as spices, condiments, vegetables, food flavours and
medicines. The resurgence of interest in herbs and the potential lucrative
anticipated revenues from the herbal industry have spurred renewed
interest in exploiting traditional knowledge and practices into scientific
realities. Current research priorities offer promising development of natural
resources into neutraceuticals, cosmeceuticals and biopharmaceuticals.
Hence the need to profile or fingerprint species for quality control and
consistency of the species utilized. It is also important to establish protocols
for micropropagation as a means of providing consistent supply of stable
and elite materials for mass propagation and commercialization. Selected
examples of indigenous uses, species profiles and successful micropropagation
of cultivated gingers are discussed.

Introduction

Besides Thailand, Malaysia represents one of the richest region in terms
of Zingiberaceous species in South East Asia. Approximately 60 % of the
total land area of Malaysia is reportedly covered by tropical rainforests
and an estimate of 15,000 species of flowering plants has been recorded. Of
these, there are more than 320 species of Zingiberaceae (excluding many
undescribed taxa) representing 21 genera. In Peninsular Malaysia, there
are roughly 160 species of Zingiberaceae belonging to 18 genera (Larsen
et al., 1999). Worldwide there are more than 1,200 species of Zingiberaceae
belonging to more than 50 genera. Hence the total count for Malaysia

72 Gard. Bull. Singapore 59 (1&2) 2007

account for at least 20 % of the world taxa and 40 % of the world genera. The
Zingiberaceae species have been utilized for various purposes worldwide
and have been a part of the Asian culture since time immemorial. In Malaysia
Zingiberaceae species are used as spice, condiment, food flavour, vegetable,
beverage, medicine, ornamental as well as in rituals associated with beliefs,
customs and traditions. Of late some cultivated gingers are exploited for the
cosmeceutical, neutraceutical and pharmaceutical industry. Between 16-20
% of the Peninsular Malaysian gingers are edible and these are consumed
fresh, cooked, pickled or boiled. The plant parts consumed are mainly
rhizomes but the inflorescences, fruits, seeds, young shoots and rarely leaves
are also utilized.

Utilization

The growth rate in global herbal industry is estimated at 7 % annually
with an estimated value of USD $183 billion in 2005. Blessed with a rich
biodiversity, Malaysia has identified the herbal industry as another source of
economic engine of growth and as such has provided the relevant national
policies for the development of this important industry. In the Malaysian
scenario, ginger has been identified as one of the 10 most popular local
herbs that have great commercial potential (data from Malaysian Herbal
Corporation). In maximising the economic potential of our rich bio-resources,
ethnic knowledge and practices need to be exploited and developed into
scientific realities. One of the significant contribution of such knowledge
is the practices of traditional complementary medicine (TCM). Global
market surveys have revealed that TCM plays a major role in the healthcare
market both in developing and industrialized nations, most prominent being
China (100%), followed by Africa (70-80%) and India (70%) (data from
Malaysian Herbal Corporation). Our ethnobotanical surveys of selected
states in Peninsular Malaysia representing East, West and South West region
of Peninsular Malaysia revealed some interesting findings with regards to
TCM.

In general, the practices of TCM in the villages surveyed are
influenced by the following factors:
® socio — economic status;
availability of modern medicine;
remoteness of the village;
availability of the herbs used in TCM;
the age of the population;
the traditional knowledge of the population.

Cultivated Gingers of Peninsular Malaysia 7

The results of our ethnobotanical surveys also indicated that
Zingiberaceae species are among the most frequently used herbs in folk-
medicine. For instance, many medicinal gingers are utilized for women-
related ailments or healthcare, such as post- partum medicine, post-natal
care treatment in the form of tonic, herbal extracts, decoctions, ointment,
aromatic herbal bath, etc. These gingers are also reported to be carminative.
Various species are used either as single plant or in herbal mixtures with
several Zingiberaceae species or other herbs for treatment of arthritis,
skin infections, inflammation, stomach-ache, muscle pains and strains etc.
Selected examples of indigenous uses are presented as follows:

1. Post partum medicine and Post-natal health care

e rhizomes of Boesenbergiarotunda(L.)Mansf.eaten raw or pickled.

e rhizome juice of Curcuma longa L. and Zingiber ottensii Valeton
drunk.

e leaves of various combinations of Zingiberaceae species, such
as Alpinia galanga (L.) Willd., Curcuma longa, Amomum
compactum Sol. ex Maton, Etlingera elatior (Jack) R.M. Sm.,
Zingiber montanum (J. Konig) A. Dietr., Curcuma mangga
Valeton & Zijp in combination with other aromatic herbs such
as Pandanus amaryllifolius Roxb., Cymbopogon nardus (L.)
Rendle etc. are boiled and used as an aromatic herbal bath for
ladies in confinement. This is practised for 2 weeks or throughout
the confinement period (42 days).

2. Dysmenorrhea
e rhizome juice of Alpinia conchigera Griff. mixed with water and
drunk.

3. Treatment for skin fungal infection (panau)
e ground rhizomes of Alpinia conchigera mixed with vinegar or
kerosene and rubbed on infected parts.

4. Treatment for jaundice
e Zingiber officinale Roscoe boiled with Alpinia galanga, Vigna
radiata (L.) R. Wilczek (mung bean), garlic and vinegar and the
decoction drunk.

5. To relief flatulence/stomachache/colic
e rhizome juice of Zingiber officinale drunk.
e rhizome juice of Curcuma zedoaria (Christm.) Roscoe drunk.
e rhizome of Curcuma mangga eaten raw with rice.
e rhizome juice of Alpinia galanga mixed with other herbs and
drunk.

74 Gard. Bull. Singapore 59 (1&2) 2007

6. Treatment for muscle pains & strains
e decoction of whole plant of Curcuma aeruginosa Roxb. drunk.
e oil of Alpinia conchigera applied topically.

7. Treatment for sprain
e poultice rhizome of Kaempferia galanga L. with rice, applied
topically.
e poultice leaves of Zingiber zerumbet (L.) Sm., applied topically.

8. Health drink/treatment for lethargy
e rhizome juice of Alpinia conchigera and fresh milk drunk in the
morning.

9. Treatment for aching joints (e.g., knee joints)
e rhizome juice of Zingiber officinale Roscoe var. rubrum Theilade
and vinegar, applied topically.
e rhizome juice of Zingiber officinale drunk.

10. Treatment for hypertension
e rhizome of Kaempferia galanga eaten raw.
e rhizome of Zingiber zerumbet eaten raw.

11. Flavour
e leaves of Elettariopsis curtisii Baker for flavouring fish dish.
e leaves of Curcuma longa for flavouring vegetable, fish and meat
dishes.
e rhizomes of Alpinia galanga, Curcuma longa, Zingiber officinale
for flavouring various dishes.

12. Cosmetic powder
e rhizome of Curcuma zedoaria ground finely with glutinous rice
and 100 types of flowers and soaked in water.
e leaves of Kaempferia galanga mixed with rice and several
aromatic plant parts, ground and soaked in water; residue used
as cosmetic powder.

Utilization: nutritional value of edible gingers

Realizing the diverse utilization of the cultivated gingers, studies have
been carried out to investigate the nutritional composition of these species.
Generally our result showed that the moisture content of the rhizomes is
high exceeding 70% and low in crude fibre content. The low crude fibre
content renders these species suitable as spices. The fat and carbohydrate
content are relatively low in the species studied (Table 1). Table 2 shows the

Cultivated Gingers of Peninsular Malaysia 75

data on thiamine, riboflavin and vitamin C of 5 cultivated gingers. Vitamin
C content is generally low except for peeled rhizome of Zingiber officinale
(11mg/100g) and young rhizome of Curcuma mangga (15.46 mg / 100g). This
may justify the consumption of the young rhizome Curcuma mangga as a
fresh vegetable in Peninsular Malaysia. Our studies on some mineral content
of Alpinia galanga, Curcuma longa, Kaempferia galanga and Etlingera
elatior (Table 3), indicated that ferum content is quite high in the roots of
K. galanga (78.30mg /100g) and in rhizomes of E. elatior (67.10mg/100g).
This result supports the development of K. galanga as a health drink as
the rhizomes and the roots are usually taken as a whole when consumed.
The screening on the anti-nutritional content showed that no cynogenic
glycosides were detected in the twelve cultivated gingers studied (Rahim et
al., 1991; Ibrahim et al., 1994).

Table 1. Proximate composition of some common Zingiberaceae species (per 100 g) [Hashim
et al., 1988; *Tee et al., 1988; **English and Lewis, 1991; ***Zanariah et al., 1997]

Energy | Moisture | Protien
Species Part
ene (g)
Young
a ‘ ‘ P
: Young
*
86.1 ‘i

: _| Mature
rhizome

sh
g)

at | Carbohydrate | Fibre
g) (g) (g)

ii)
—

s}o|o Solu S

~Jj}~ Jj SJ (Nn SS -
~]

v2 a hs [Ear [ao oo | #
(oe) oe (o-e) \O —

ioe)

—

A. galangal***|Rhizome 71 | 81.5_|

Young |
Emme face] a

13.0

0. 12

— —S 2 — oe) — —
— ON | ON J Go ~] N ~ ~]
=) Iolo n =) o
Nn ByBR] OU No ~ Nn

*
1 Gy
au
Ss
3
san)
a
=
D
Q
N
©
if
<q
1oS)
a
(oe)
: so : :
oS)
N —
oN oe) ~]

76 Gard. Bull. Singapore 59 (1&2) 2007

Table 2. Vitamin composition of some Zingiberaceae species (mg/100g weight) [Hashim er
al., 1988; *Tee et al., 1988; **English and Lewis, 1991; ***Zanariah et al., 1997]

[Species [Pant | Thiamine [Ribo flavin] Ascorbic acid
Z. officinale

I barrie aa?)
|Z. officinale* | Youngrhizome}| 0.04 | 0.04
i | 0.06
as OO
I hag fi Sane
04

Sines
0
LC domestica | Fresh thizome [~~
[Z zerumbet [Young thizome | —
0.40
001
Young thizome[ = [=

Ginger products

.04
.04
.03
.03

The popularity of herbal products has increased greatly in recent years due
to the comsumer’s preference for natural ingredients in their medicine, food
and personal care products. Several cultivated gingers in particular, Zingiber
officinale, have been developed commercially (globally and locally) into
various herbal products. Selected examples are given below:

1. Medicine
e Arthritis (e.g., Zinaxin).
Nausea —tablets.
Anti-inflammatory products.
Flatulence and indigestion (e.g., Eno).
Post partum medicine including the Indonesian jamu (capsules,
tablets, tonic, powder, poultice).
e Medicinal oils / ointment.
e Balm.

2. Health products
e Ginger and ginseng capsules.
e Ginger and garlic capsules.
e Dietary supplement (vitamin C supplement).

rie

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78 Gard. Bull. Singapore 59 (1&2) 2007

Herbal teas.
Functional beverages.

3. Personal care

e Aromatic soaps.

e Aromatheraphy products (e.g. massage oils, scrubs, bath oils, etc.).
e Perfumes.

e Shampoo and conditioner.

e Various cosmetics.

4. Skin care
e Mosturizers and toner.
e Skin lightening/whitening cream.
e Anti-aging/anti-wrinkle cream.
e Anti-acne cream.
e Anti-eczema cream (e.g., Psoriosis).

. Food, confectionery and sweets
e Biscuits / cookies/cakes.
e Ice cream.
e Chocolates and sweet preserves.
e Jam, chutney.

Profiles

In developing plant based products, research and quality control
measures on the potential natural resources need to be intensified and
established. In this respect, species profiling is considered as a useful tool
in maintaining quality, especially when there are problems of presence of
adulterants. Several techniques of species profiling are usually used such
as chemical profiling, DNA fingerprinting and other botanical techniques.
Our studies have revealed that DNA fingerprinting technology is useful
in authentication of not only species but also varieties and variants (Plate
1 and Plate 2). For instance, based on RAPD primer OPA 4 as shown in
Plate 1, Zingiber zerumbet and its three variants could be differentiated
quite easily. In another study (Plate 2), local varieties of Zingiber officinale,
namely Zingiber officinale var. rubrum (halia padi) and Zingiber officinale
var. rubrum (halia bara), were shown to differ very slightly in their DNA
profiles based on RAPD primers OPA 1, OPA 8 and OPA 20.

Some botanical methods are also useful in identifying species,
such as anatomy of leaves and petioles and SEM studies on reproductive
structures of plants. These botanical techniques are useful as ancillary tools

Cultivated Gingers of Peninsular Malaysia 79

| Lanes

fee = Zingiber zerumbet
4 & 5 — Zingiber zerumbet (v1)
ee Tc Zingiber zerumbet (v2)
18 &9- Zingiber zerumbet (v3) |
i 10 & 11 — Curcuma zanthorrhiza
| |

Plate 1. DNA Profile of variants of Zingiber zerumbet and Curcuma zanthorrhiza (RAPD
primer OPA4).

—
—_
! —
:
:
| —
| ae

1) eee

Ps —

Plate 2. DNA profiles of Zingiber officinale and its varieties (RAPD primers OPA1, OPA8
& OPA20).*X’ shows the polymorphic banding patterns distinguishing all varieties studied;
(OPA1: X=1500bp, OPA8: X=2000bp, OPA20: X1=1350bp, X2=900bp, X3=800bp, X4=550bp)
H = Z. officinale Rosc var. officinale (halia), HB = Z. officinale var. rubrum (halia bara), HP
= Z. officinale var. rubrum (halia padi), M= Marker 100bp Ladder Plus.

in authentication of species used in product development. Plates 3, 4 and 5
exhibit clearly the differences between selected gingers in their transverse
sections of leaf midribs, petioles and margins respectively. Although SEM
features of pollen, stigma and labellum of some gingers may not be as
distinct, in most cases these data are also useful in species identification as
shown in Plate 6.

80 Gard. Bull. Singapore 59 (1&2) 2007

2 i 2
+ 95--s ee
— i

‘ozs
ate.

F) K. pulchra G) K. rotunda

Scale bars = 500 um (A,B,C)
Scale bars = 200 um (D,E,F,G,H)

Plate 3. Transverse sections of leaf midribs.

Cultivated Gingers of Peninsular Malaysia

K. pulchra

Scale bars = 500 um

Plate 4. Transverse sections of petioles.

K. rotunda

82 Gard. Bull. Singapore 59 (1&2) 2007

I) K. pulchra J) K. rotunda

Scale bars = 200 um (A,B,C,D,E)
Scale bars = 50 um (F,G,H,I,J)

Plate 5. Transverse sections of leaf margins.

Cultivated Gingers of Peninsular Malaysia 83

Pollen

Be0083 6KY
Boesenbergia plicata Scaphochlamys kunstleri |§ Hedychium coronarium

Stigma

| — 160Vm-

Boesenbergia rotunda Kaempferia galanga Scaphochlamys klosii
Labell

aa ae ate i

Boesenbergia plicata Boesenbergia rotunda Hedychium coronarium
(Middle labellum) (Middle labellum) (Lower labellum)

A, B, C (x 10um)
D, E, F (x 100um)
G, H, I ( x 100um)

Plate 6. SEM studies of flower parts.

84 Gard. Bull. Singapore 59 (1&2) 2007

Micropropagation

One of the common problems in the development of herbal products is the
sufficient supply and consistency of the source materials. Micropropagation
by tissue culture technique is a means of providing consistent supply of stable
and elite materials for mass propagation. For the last ten years, our team
has managed to establish protocols for micropropagation of at least fifteen
cultivated gingers most of which are of medicinal importance. In general,
an average of 3-5 shoots per explant were successfully regenerated on MS
medium supplemented with 3.0 % (w/v) sucrose, 0.2 % (w/v) phytagel and
1.0 —- 3.0 mg/L BAP (Plate 7).

ae a

Production of multiple shoots

Plate 7. Micropropagation of Zingiber officinale and its varieties

Our field studies on the in vitro and normal plants of Zingiber offici-
nale and its varieties showed that after transplanting, the field performance
of in vitro plants were found to be superior compared to the control plants
based on five quantitative traits as stated in Table 4. This result implicates the
significance of establishing micropropagation protocols for the sustainable
utilization of commercially important herbs such as the cultivated gingers.

Cultivated Gingers of Peninsular Malaysia

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86 Gard. Bull. Singapore 59 (1&2) 2007

Acknowledgements

The authors thank the Ministry of Science, Technology and Environment
of Malaysia for the major research grants and University of Malaya for the
short termed research grants. The authors wish to thank all collaborators,
students and technicians involved in the various projects.

References

English, R. & J. Lewis, J. 1991. Nutritional values of Australian foods. Cited
in Zanariah et al. (1977). Journal of Tropical Agriculture and Food Science
25(2): 225-229.

Hashim, H.M., H. [brahim and Z.H.A. Rahim. 1988. Preliminary studies on
some nutritional composition of the edible gingers, pp. E1-2. In: A. Sipat
et al. (eds.). Advances In Biochemistry And Biotechnology In Asia And
Oceania. Kuala Lumpur, Malaysia.

Hussin, K.H. and H. Ibrahim. 1989. Taxonomic implications of several
Zingiber species (family Zingiberaceae) based on morphological and
anatomical characteristics. Malaysian Applied Biology 18: 155-161.

Hussin, K.H., T.S. Chuah, H. Ibrahim, O.G. Wu, J.P. Liao and N. Liu. 2000.
Comparative leaf anatomy of Alpinia Roxb. (Zingiberaceae) from China.
Botanical Journal of Linnean Society 133: 161-180.

Hussin, K.H., H. Ibrahim, H.A. Ali D. Aminah, J.P Liao and N. Liu.
2001. Anatomical variations in leaves of Boesenbergia O. Kuntze and
Kaempferia L. (Zingiberaceae). Journal of Tropical and Sub-tropical
Botany 9: 49-54.

Ibrahim, H. & Z.H.A. Rahim. 1988. Distribution of K, Na, Ca, Mg, Fe, and
Zn in four species of the Zingiberaceae. Malaysian Journal of Science 10:
25-29.

Ibrahim, H., S. Mohd. Nor and Z.H.A. Rahim. 1994. Medicinal gingers
(Fineiheresss) - analysis of oxalate, tannin and cyanogenic glycosides,
pp. 125-127. In: S.H. Ton et al. (eds). Proceedings of the 19" Malaysian
Biochemical and Molecular Biology Society Conference. Bangi,
Malaysia.

Cultivated Gingers of Peninsular Malaysia 87

Ibrahim, H., H.C. Ong and R. Hassan. 2000. Ethnobotanical survey of the
ginger family in selected Malay villages in Peninsular Malaysia. Malaysian
Journal of Science 19: 93-99.

Larsen, K., H. Ibrahim, S.H. Khaw and L.G. Saw. 1999. Gingers of Peninsular
Malaysia and Singapore. Natural History Publications (Borneo), Kota
Kinabalu. 135 pp.

Mohd. Azmi, M., N. Khalid and H. Ibrahim. 1999. Pendekatan baru dalam
propagasi spesies-spesies halia ubatan terpilih. Transaction of Malaysian
Society of Plant Physiology 8: 55-58.

Muda, M.A., N. Khalid and H. Ibrahim. 2004. Micropropagation study on
three varieties of Zingiber officinale Rosc. Malaysian Journal of Science
23(2): 7-10.

Muda, M.A., H. Ibrahim and N. Khalid. 2004. Differentiation of three
varieties of Zingiber officinale Rosc. by RAPD fingerprinting. Malaysian
Journal of Science. 23(2): 135-139.

Noraini, T., H.H. Khatijah and H. Ibrahim. 2005. Comparative Leaf Anatomy
of Alpinia Roxb. species (Zingiberaceae) in Peninsular Malaysia. Nordic
Journal of Botany 23: 463-483.

Nordiana, M.,M. Yusoff and H. Ibrahim. 1997. Scanning electron microscope
study on selected species of the tribe Hedycheae (Zingiberaceae), pp.
251-253. In: A.R. Razak et al. (eds.). Proceedings of the First ASEAN
Microscopy Conference. Johor, Malaysia.

Nordiana, M., M. Yusoff and H. Ibrahim. 1998. Ultrastructure of leaf surfaces
of Scaphochlamys spp. (Zingiberaceae), pp. 118-121. In: Proceedings of
the 7" Scientific Conference of the Electron Microscopy Society Malaysia.
Ipoh, Malaysia.

Nordiana, M., H. Ibrahim and M. Yusoff. 2000. Floral characteristics of
Boesenbergia O. Kuntze., Kaempferia Linn. and Scaphochlamys Bak., pp.
215-217. In: Proceedings of the 9" Scientific Conference of EMSM 2000.
Malaysia.

Rahim, Z.H.A., H. Ibrahim and H.M. Hashim. 1991. Edible gingers
(Zingiberaceae family) — determination of tannin, oxalate and cyanogenic
glycoside content, pp. 183-187. In: A. Wan Azlina (ed.). Proceedings of

88 Gard. Bull. Singapore 59 (1&2) 2007

the 16" annual conference of the Malaysian Biochemical Society. Johor,
Malaysia.

Tee, E.S., N. Mohd. Ismail, A. Mohd. Nasir and I. Khatijah (compilers). 1988.
Cited in Zanariah et al. (1977). Journal of Tropical Agriculture and Food
Science 25: 225-229.

Zanariah, J., A. Noor Rehan and O. Rosnah. 1997. Nutritional composition
of common Zingiberaceae species used in traditional medicines and
cooking. Journal of Tropical Agriculture And Food Science 25: 225-229.

Gardens’ Bulletin Singapore 59 (1&2): 89-104. 2007 89

An Introduction to the New Guinea Database,
with Notes on the Zingiberaceae,
Specifically Riedelia Oliv.

R. JOHNS

Botanical Research Institute of Texas [BRIT], 509 Pecan St., Fort Worth, Texas,
46102 — 4060, U.S.A.
Email: mountjayal @yahoo.com

Abstract

The entries for the family Zingiberaceae in the New Guinea database include
records of over 1700 collections. Based on this information an overview of
the family is presented. The extensive records in the database highlight
several problems. A considerable number of collections in the family have
not been identified at generic level (190 collections) and many collections in
each genus are not identified at the species level. This particularly applies to
the larger New Guinea genera: Alpinia Roxb. and Riedelia Oliv., respectively
with 256 and 236 collections. In this paper particular attention is paid to the
neo-endemic genus Riedelia, which is represented by 85 described taxa in
New Guinea. The genus has not been revised since 1916.

Introduction

The objective of the New Guinea program at BRIT is to instigate a period of
intensive botanical collecting of the flora through expeditions to both Papua
and Papua New Guinea. These expeditions will be carried out in association
with staff from National and Regional Herbaria. It is essential to collect
‘high quality’ herbarium materials, photographs, and living collections for
the scientific study of the flora. The top set of collections from New Guinea
will be deposited locally in the major National herbaria with duplicates sent
to important international herbaria. Only this will provide an adequate basis
for future studies of the flora of New Guinea.

A database is being developed to facilitate the study of the flora of
New Guinea. It will not only enable the mapping of plant distributions but
will also highlight areas where the flora is unknown or poorly know and
will thus act as a guide for future expeditions. The database will also enable
the production of species lists for given regions. Another benefit will be to

90 Gard. Bull. Singapore 59 (1&2) 2007

provide information for younger botanists and ecologists, for the study of
plant diversity in New Guinea and also to access the major literature on the
New Guinea flora. The database, which so far includes ca 300,000 herbarium
collections, has considerable potential for use in herbarium management. It
will also be an essential tool to identify conservation areas and to highlight
biodiversity priorities for all levels of government in New Guinea.

1. The New Guinea database

The New Guinea database includes collections from the Indonesian
Province of Papua, previously Irian Jaya (now divided into two Provinces)
and Papua New Guinea. Because of its floristic associations with New
Guinea, collections from the Aru (Aroe) Islands (Maluku Province) are
included. Records are also included from the North Solomons Province
(Buka and Bougainville), politically, part of Papua New Guinea. Extension
of the database to the Solomon Islands would include all records for the
biogeographical region known as Papuasia.

The New Guinea database 1s arranged to include:

A species database. The type collection of each species is recorded. The
herbarium in which the type occurs is also recorded (holotype and isotypes
where known), its place of publication, important literature, and general
notes on each species and their ecology. The distribution is recorded and,
where the species is endemic to New Guinea, this is recorded as a regional
or a local endemic. A note is included if there are obvious nomenclatural
problems. Synonyms are also recorded.

A specimen database. It is proposed to list all vascular plant collections
from New Guinea in the database. So far nearly 300,000 records of vascular
plants have been entered into the database(s). Each collection will be geo-
referenced so that its location can be mapped.

Using the entries in the database it will be possible to produce
distribution maps of expeditions and collections at the family, genus and
species levels. This will be a valuable guide so that areas, which have been
poorly collected, can be targeted for expeditions. Locality data will also
enable the generation of species lists at provincial and sub-provincial levels
as a basis for regional planning and conservation. From the database it will
be possible to produce:

a) Lists of all plant species known from New Guinea, the Provinces
and smaller regions within New Guinea;
b) Lists of species from selected plant families, and genera. It will

New Guinea Database on Zingiberaceae 91

also be possible to map these.
c) Lists of plants endemic to the New Guinea region, and to map
their patterns of distribution.

2. The family Zingiberaceae in New Guinea

The New Guinea region is one of the major centres of diversity for the family
Zingiberaceae. Larsen et al. (1998) give the diversity of the family, worldwide,
as ca 52 genera and 1,300 species. Based on the estimates of Newman
(2007), New Guinea probably has over 300 species. In total New Guinea
includes over 20 percent of the species worldwide. Over 1700 collections
of Zingiberaceae have been databased from New Guinea; the database
includes records of many species new to science. The database includes 240
species in 18 genera from New Guinea (Table 1). Newman (2007) lists six
major genera from New Guinea: Alpinia Roxb., Amomum Roxb., Etlingera
Giseke, Hornstedtia Retz., Pleuranthodium (K. Schum.) R.M.Sm., and
Riedelia Oliv. He also lists several genera for New Guinea, which he thinks
are cultivated or possibly naturalised, these include Curcuma L., Globba
L., and Zingiber Mill. Many of these genera have ‘endemic’ species listed
from New Guinea (Newman et al., 2004). Their status will require detailed
taxonomic studies as all genera and species are poorly studied. Potentially
many new species remain to be collected and described from the large areas
that are still un- or under-collected. In Table 1 the number of collections in
a genus, percentage of endemism, and the number of species recorded from
Papua, Papua New Guinea and the whole island is enumerated for selected
genera. These figures will change as our knowledge of the family in New
Guinea increases.

3. History of research on Zingiberaceae in New Guinea

The Zingiberaceae in New Guinea were revised in several important papers
by Schumann (1899, 1904), Valeton (1913a, b, 1914, 1917), and Ridley (1886,
1916, 1923). Few general papers have been published since the work of
Valeton and Ridley. The recent works of B.L. Burtt and R.M. Smith are
the first modern attempt to examine the family in Malesia (Burtt & Smith,
1972a, b; Smith, 1975, 1990a, b,c).

To date, the family in New Guinea has received little attention. With
the exception of Alpina, Pleuranthodium, and Riedelia, the family is poorly
represented in New Guinea. Of the 223 species recorded from New Guinea
by Newman et al. (2004), 90 taxa are referred to Riedelia and 46 to Alpinia.

4. The collections of Zingiberaceae in New Guinea
In Papua the majority of species are described from several key areas (Fig. 1).
The Kepala Burung (= Vogelkop or Bird’s Head, see Fig 1A). L.S. Gibbs

92 Gard. Bull. Singapore 59 (1&2) 2007

Table 1. Collection data on selected genera of Zingiberaceae from New Guinea.

No of No of Endemism % Species Recorded

taxa collect. ' Pap. PNG Both
Alpinia 63 473 [256] 3 96 alas 2 8
Amomum 14 Ol 130) 92 6 7 1
Curcuma 6 73 [20] 57 3 1 3
Etlingera 16 69 [23] 100 8 6 2
Globba 1 9 [4] 0 - 1 -
Guillainia 4 18 [-] 100 1 - -
Hornstedtia 5 108 [24] 66 Z - 1
Pleuranthodium 2p 63 [10] 100 5 14 3
Riedelia 85 686 [251] 100 a2 il (Ziad
Zingiber 6 19 [-] 50 1 2 3

Zingiberaceae indet. 191?

Totals 231* a reef

‘Total number of collections in the category.
* Number of collections not identified to genus.
3[ ] Number of collections not identified to species level.

Nine species have be added from database records for ‘doubtful’ genera in Papua New
Guinea:

Hedychium [1 species - H. coronarium|

Phaeomaria [2 species - P. anthokaphina, P. novoguineensis]|

Thylacophora {1 species - T: pognocheila]

Naumannia [1 species - N. insignis (Riedelia insignis)|

In addition Riswan and Setyowati (1996) listed Kaempferia (3), and Nanochilus (1) from
Papua. Pleuranthodium (Psychanthus) is also not listed by Riswan and Setyowati (1996)
from New Guinea.

organised an expedition to the Arfak Mountains in 1913-1914. Gjellerup
made additional collections of gingers from the area in 1912. These ginger
collections were described by Valeton in Gibbs (1917). More recent
expeditions to this area contain many collections that have not been critically
studied. These include collections by staff of the Forestry Department (BW
series), collections by P. van Royen and H. Sleumer from the Kebar Valley,

New Guinea Database on Zingiberaceae 93

and the expeditions to the N.E. Kepala Burung, funded by the MacArthur
Foundation, which were made in the 1990's. Staff from Herbarium Bogoriense
and the Manokwari Herbarium have also collected in the area.

The other key areas in Papua are Mt Jaya (Carstensz) (Fig. 1B) and
the Lorentz River Basin (Fig. 1C), both to the S of the central Ranges. C.B.
Kloss was the botanist/collector for both of the Wollaston Expeditions to
Mt Carstensz. The second expedition resulted in the collection of many
‘new’ species of gingers (Ridley 1916). Some of these species have been
recently recollected by the staff of Freeport (Environmental Department)
and also during the recent expeditions to Mt Jaya by expeditions organised
from the Royal Botanic Gardens, Kew (Johns et al. 2006). Collections have
also been made in the same area by staff from Herbarium Bogoriense and
the Manokwari Herbarium. The Lorentz basin was collected by several
expeditions and many species were described by Valeton (1913b). The main
collectors were Lorentz, von Roemer, and Versteeg. Some species have
also been described from the early collections in the vicinity of Jayapura (=
Hollandia, see Fig. 1D). The extensive collections from Papua by H.J. Lam
in 1920-1921, and L.J. Brass in 1938-1939, and collectors in the BW series all
postdated the most recent revisions of the gingers from Papua.

The history of Zingiberaceae research in Papua New Guinea parallels
that of Papua. In 1875 Nauman collected in the Bismark Archipelago (Fig.
1L) as did several latter collectors, including Nymann in 1899 and Peekel
from 1908 to 1938. In 1885 Forbes made extensive collections (Ridley 1886)
from the Sogeri Division (Fig. 11), which included many Zingiberaceae. The
last collections from Papua New Guinea studied by specialists in the family
(Valeton 1914) were those of Schlechter in 1909 from the Toricelli Mts. (Fig.
1E) and various collectors in the Madang and Morobe Provinces (Fig. 1F,
G, H). There have been extensive collections from Papua New Guinea since
1916, especially in the Central Highlands (Fig. 1J) and the upper reaches
of the Fly River (Fig. 1K) but all postdated any specialist studies of the
Zingiberaceae.

In common with many plant families in New Guinea the species
of Zingiberaceae are very poorly known. While many tree species have
been widely collected (both lead herbaria in New Guinea were part of the
Forestry Departments; Papua — BW series; PNG — NGF and LAE series),
the majority of herbaceous species, including the gingers, are poorly known.
As shown in Table 2 most species of gingers are known from only the types,
or two to three collections. This is also characteristic of many herbaceous
plant families in New Guinea, where over sixty percent of the species are
often represented by a single collection. Another fifteen to twenty percent
of species are usually represented by only two to three specimens.

Gard. Bull. Singapore 59 (1&2) 2007

Figure 1. Critical sites to be visited for the recollection of the duplicates of types of Riedelia

in New Guinea. For location of sites see text.

Bismarck Archipelago ; S
New Irela
» j >
. ae : Qq ‘
g New Britain SI
H

St Matthias
Manus $ * .
— ri
: mS Tabar

Table 2. Number of specimens collected (1-10+) per species from selected genera of

Zingiberaceae from New Guinea.

Collections 1 2 3 6 7 8 9 10+
per species

Alpinia AG» (4 AS 46H 1 - - 1 3
Amomum Ly gees - - - - - :
Curcuma + 1 - - : a = fi
Pleuranthodium

(Psychanthus) i: 8S 6 - - 1 - -
Riedelia la ale 2 - - vé 8
Zingiber 2 - - 1 1 - - -

5. The genus Riedelia nom cons. in New Guinea and Maluku

The genus Riedelia was described by Oliver in Hooker's Icon. Pl. 15: 15
(1883), based on acollection by J.G.F. Riedel from Pulau Buru in the Maluku.
The type species is Riedelia curviflora (Plate 1). Riedelia was conserved
against Riedelia Cham. described in the Verbenaceae in 1832, while another

New Guinea Database on Zingiberaceae 95

homonym Riedelia Meisn., a Brazilian genus described in Ericaceae in
1863 was not validly published (Rickett & Stafleu, 1959). Valeton (1914)
incorrectly lists Riedelia curviflora Oliv. as a synonym of Riedelia lanata
(Scheff.) Valeton, a widespread species from both Papua and Papua New
Guinea.

Newman et al. (2004) list two genera as synonyms of Riedelia,
Nyctophylax Zipp. described by Zippelius in 1829, based on N. alba Zipp.
a nomenclatural synonym (Greuther ef al. 2000) and Thylacophora Rid.
(1916) based on T. pogonocheila Ridl., collected by C.B. Kloss from Mt
Carstensz (Mt Jaya) in 1912. Hedychium lanatum Scheff. was described in
1876 based on a collection by J.E. Teysmann (Teysmann 6741), collected
from the MacCleur Gulf in New Guinea in August of 1871.

The monotypic genus Naumannia Warb. [| Bot. Jahrb. 13: 452 (1891)]
was based on N. insignis. The genus was distinguished by its petaloid lateral
staminodes and the flower lacks a labellum. It is possibly related to R. cor-
allina. Further collections are required from the type locality, Sattelburg,
Morobe Province, to establish its status. Readers should also note that the
status of Nyctophylax alba Zipp. (1829), the first record listed as a synonym
of Riedelia by Newman et al. (2004), is at present not understood.

The publication of the 90 taxa (species and varieties) of Riedelia from
New Guinea is summarised in Table 3. The major papers were published by
K. Schumann (1904) and a series of papers by Valeton (1907, 1913a, 1913b,
1914, 1917). Ridley (1916) described the collections made by C.B. Kloss
from Mt Carstensz (Mt Jaya). There have been no detailed studies of the
genus Riedelia since the publications of Valeton and Ridley. In 1979 P. van
Royen published five species from the subalpine and alpine regions of New
Guinea, and A. Gilli (1983) published two new species from the highlands
of Papua New Guinea (Table 2).

6. Distribution of Riedelia

The genus Riedelia is distributed from the Maluku to New Guinea, extending
to the Solomon Islands (Bougainville) and is represented by ninety taxa
in New Guinea (Fig. 2). Of the 686 collections of Riedelia, 251 still remain
unidentified. All species are endemic to New Guinea, but R. curviflora
possibly extends its range to Papua from the Maluku. Forty-two species are
known to be restricted to Papua, 32 species to Papua New Guinea, and 11
species occur throughout the island. A more detailed understanding of their
distribution patterns must await more detailed study.

Species identified as Riedelia are also recorded from Borneo
(Sarawak [Ashton 17713; Meijer 21217], and Kalimantan [Kostermans
5122]), Sulawesi [Burley 3511, 36671], and the Philippines (Newman, pers.
com.). The taxonomic identity of these collections requires further study.

96 Gard. Bull. Singapore 59 (1&2) 2007

372

Fig. i ie oS
‘6 44. A—C Riedelia curviflora Oliv. A Inflorescentia. B Flos. B Gi

(A Icon sec. Oliver reiterata, reliquae originariae).

Plate 1. Riedelia curviflora Oliver. A. Inflorescence; B. Flower; B’. Calyx; C. Fruit. A-C from
Riedel s.n., Pulau Buru, Maluku. Reproduced from: Schuman, 1904.

New Guinea Database on Zingiberaceae 97

Table 3. Publication dates for the species of Riedelia [86 taxa]. Source of Type collection: °
Moluccas, ! Papua, ? Papua New Guinea

Date
1883

1904

1907

Author
Oliv.

K. Schum.

Valeton

1913a Valeton

1913b Valeton

1914

1916

LOT

1923

1979

1983

1990

Valeton

Ridley

Valeton

Ridley

P. Royen

Gilli

R.M. Smith

Publication

Hooker’s Icon. Pl., Pl. 15:15. [1 taxon] R. curviflora° [Type species for
genus].

Pflanzenr. IV, 46: 373-440. [7 taxa] R. affinis (Ridl.) K. Schum.?,
R. albertisii (K. Schum.) K. Schum.?, R. bismarckii-montium?, R.
macrantha (K. Schum.) K. Schum.!, R. monophylla?, R. nymanii*, R.
stricta?

Bull. Dép. Agric. Indes Néerl. 10:2. [1 taxon] R. geanthus !

Icon. Bogor. 4(3): t. 373-375. [3 taxa] R. corallina (K. Schum.) Valeton
2, R. erecta ', R. lanata (Scheff.) Valeton !

Nova Guinea 8: 962-980. [30 taxa] R. alata ', R. angustifolia ', R.
areolata !, R. arfakensis !, R. brachybotrys', R. branderhorstii ', R.
brevicornu ', R. epiphytica !, R. eupteron ', R. fulgens ', R. graminea },
R. graminea var diversifolia ', R. graminea var. elata', R. graminea vat.
nana ', R. hollandiae ', R. lanata (Scheff.) Valeton forma ligulata ', R.
macranthoides 1, R. maculate }, R. maxima '!, R. montana !, R. montana
var. arfakensis 1, R. montana var. golianthensis |, R. orchidoides
(K. Schum.) Valeton !, R. paniculata |, R. pterocalyx ', R. robusta |,
R. sessilanthera ', R. sessilanthera var. euodon ', R. subulocalyx 1, R.
tenuifolia |

Bot. Jahrb. Syst. 52: 70-96. [21 taxa] R. bidentata *, R. decurva (Ridl.)
Valeton *, R. dolichopteron 7, R. ferruginea 7, R. flava Lauterb. ex
Valeton 2, R. geluensis *, R. geminiflora 7, R. grandiligula ?, R. latiligula
2, R. longifolia *, R. longirostra *, R. macrantha var. grandiflora *, R.
macrothyrsa2, R. microbotrya?, R. minor, R. monticola?, R. rigidocalyx
Lauterb. ex Valeton 2, R. schlechteri 2, R. umbellate 2, R. urceolata 2, R.
urceolata var. sessilifolia ”

Trans. Linn. Soc. London, Bot. 9: 222-226. [11 taxa] R. aurantiaca ',
R. bicuspis }, R. ferruginea ? (nom. illeg.), R. hirtella ', R. klossii 1, R.
ligulata', R. longisepala', R. pulcherima ', R. purpurata ', R. triciliata ',
R. wollastonii !

see Gibbs, Fl. Arfak Mts. 102. [2 taxa]
R exalata ', R. montana var. puberula !

Proc. Roy. Soc. Queensland 34: 19. [2 taxa]
R. lanatiligulata 2, R. whitei

The Alpine Flora of New Guinea 2: 860-866. [5 taxa]
R. curcumoidea ', R. marafungensis *, R. rosacea ?,
R. subalpina 2, R. suborbicularis 2

Ann. Naturhist. Mus. Wien, B 84: 46-47 (“1980”). [2 taxa]
R. capillidens 7, R. geluensis Valeton var. microflora Gilli 2

Edinburgh J. Bot. 47: 65. [1 taxa]
R. cordylinoides (Ridl.) R.M. Sm.!

98 Gard. Bull. Singapore 59 (1&2) 2007

2 (11): 13
24

53 (151): 148

299

43 (328): 186
425

Figure 2. Distribution and diversity of Riedelia. Distribution of Riedelia ( ------ ).
Diversity of Riedelia in Maluku, Papua and Papua New Guinea expressed as:

No. of species (no specimens) : No. indet collections
Total number collections

7. Ecology of Riedelia in New Guinea

Most species of Riedelia grow as terrestrial species in the rainforest from
sea level to 3,200 to 3,700 m. Data is presented in Table 4 to show the
altitudinal zonation of the common genera of Zingiberaceae from New
Guinea. The three most common genera in New Guinea, namely, Alpinia,
Pleuranthodium, and Riedelia, occur at most altitudes up to 3,700 m (Table
5). At their upper altitudes species can be locally common in subalpine
forest. Most collections however do not have the altitude recorded.
Although both Alpinia, and Pleuranthodium are recorded above 3,500 m
altitude, both have fewer collections above 1000 m. Riedelia in contrast is
more strongly represented by collections from the mid- and upper montane
forests, particularly so in PNG. It is difficult to interpret these Tables as they
may merely reflect collection patterns rather than actual zonation patterns.
Higher altitude forests are more accessible in PNG than in Papua. Mid-
montane forests, being wetter than the lower montane forests, are probably
more suitable for members of the genus.

The lowland tropical rain forest is diverse with many species of
Riedelia including R. albertisii, R. aurantiaca, R. brevicornu, R. erecta, R.
geanthus, R. grandiligula, R. longifolia, R. macranthoides, R. maxima, R.
nabirensis, R. pterocalyx, R. stricta, R. subulocalyx, and R. tenuifolia.

New Guinea Database on Zingiberaceae 99

As stated above, many species of Riedelia grow in the montane zone.
Species have been subjectively allocated to a vegetation type based on the
altitude of the collections. Due to the Massenerhebung effect resulting in
vegetation zones being lower on smaller mountain masses, the simple use of
altitude as a guide to the vegetation type has probably led to inaccuracies.
The vegetation data on labels of collections of Riedelia is inadequate.
Probably several of the ‘lower montane’ species will, with more detailed
study, be placed as largely mid montane in their distribution ecology.

The following species of Riedelia have been recorded from the
lower montane zone: R. affinis, R. bicuspis, R. dolichopteron, R. decurva, R.
ferruginea, R. fulgens, R. klossii, R. ligulata, R. longisepala, R. macrantha, R.
monophylla, R. pulcherima, R. robusta, R. umbellata, and R. urceolata.

The mid-montane forest is probably more diverse than the lower
montane forest. Here, species of Riedelia include R. arfakensis, R. bidentata,
R. capillidens, R. geluense, R. hirtella, R. hagenti, R. monticola, R. orchioides,
R. paniculata, R. purpurata, R. rosacea, R. schlechteri, R. sessilanthera, R.
triciliata.

The upper montane and subalpine forests are also diverse. Species
of Riedelia known from these forests include R. bidentata, R. curcumoidea,
R. exalata, R. macrantha, R. marafungensis, R. microbotrya, R. montana,
R. monticola, R. rosacea, R. suborbicularis, and R. subalpina. The family
is represented at its highest altitudes by Riedelia montana Valeton var.
montana (Johns 2006), and by Riedelia montana Valeton var. goliathensis
Valeton (Newman, in press).

The altitudinal database includes several problematic species too,
which will require detailed study. Probably the species identifications
are incorrect on herbarium sheets. These species include Riedelia
corallianae (60-500 m and 1800-2250 m), R. epiphytica (40-1000 m
and 2200-2750 m), R. graminea (250-900 m and 2400-3460 m), and R.
lanata (10-90 m and 1500-1900 m). Another problematic species is R.
macrantha, which is collected over an altitudinal range from 10 to 3660 m.

8. Brief notes on other genera of Zingiberaceae in New Guinea
To recapitulate, the basic information on species number, number of
collections (including collections not identified), percentage of endemism,
and the distributions in Papua, Papua New Guinea and number of species
recorded from both regions is given in Table 1. Notes are included only on
the more poorly known genera. Several genera have many undescribed
species in New Guinea.

Boesenbergia Kuntze is known from a single sheet from New Guinea
collected in the Central Highlands of Papua New Guinea. W. Vink 16553 was
collected in secondary forest at 1960 m from the Minj-Nona Divide in the

100 Gard. Bull. Singapore 59 (1&2) 2007

Table 4. Altitudinal zonation of collections from herbarium specimens of selected genera
of Zingiberaceae from New Guinea. Altitudinal Zones: 1. 0 — 499 m; 2. 500 — 999 m; 3. 1000 —
1499 m; 4. 1500 — 1999 m; 5. 2000 — 2499 m; 6. 2500 — 2999 m; 7. 3000 — 3499; 8. 3500 m+.

1 2 3 4 5 6 7 8
Alpinia 4 + + + + + + 4
Amomum + + - - 5 : . x
Boesenbergia - - - - : 4. : :
Curcuma + + + - “ - .
Etlingera + + + + - " .
Globba + - - . F 6 .
Guillainia - + - : . 3 :
Hedychium + - - . : 7
Hornstedtia + + + - _ : r ‘
Pleuranthodium + + + : 4 é 3: rd
Riedelia + + + + + a ~ ~
Thylacophora + : - r - E 4 5
Zingiber + - : : : 2 : L

Table 5. Altitudinal zonation of Alpinia, Pleuranthodium, and Riedelia. The number of
collections is recorded for each altitude. (a) Collections with no recorded altitudes. Altitudinal
Zones: 1. 0 — 499 m; 2. 500 — 999 m; 3. 1000 — 1499 m; 4. 1500 — 1999 m; 5. 2000 — 2499 m; 6.
2500 — 2999 m; 7. 3000 — 3499; 8. 3500 m+.

All Zingiberaceae 599 =: - 3920 96-8 cn LO5¢ LSO | AA, pet Ot) Pee

Alpinia

Papua 65 DO. eg hdl 7 4 6 - - -
Papua New Guinea 82 2A AZ 2 2 4 - 4 1
Pleuranthodium

Papua 3

Papua New Guinea 28 5 1 1 - 1 - 3 1
Riedelia

Maluku ii 14 y i) Z 2 z bs F

New Guinea Database on Zingiberaceae 101

highlands of Papua New Guinea. Vink noted it was planted but according to
field notes ‘it was not introduced by Europeans’. It was named Singa Manga
in the Loowi Language of Papua New Guinea.

As presently known, Pleuranthodium is endemic to New Guinea, but
collections from C Sulawesi and southern Philippines could extend its range.
At present 22 species are known (Smith, 1991). Sixty three collections are
entered in the database of which 11 have not been identified. All species are
endemic. Only five endemics are known from Papua, fourteen from Papua
New Guinea, and three species occur throughout New Guinea. With the
exceptions of Pleuranthodium piundaundensis (P. Royen) R.M Sm., which
has nine collections, all other species are known from only 1-4 collections.

Conclusions

There are many problems in the study of the Zingiberaceae in New Guinea.
The family is very poorly collected from most areas in New Guinea; indeed,
large areas are uncollected. As botanical explorations proceed we can
expect a large increase in the number of species, as many appear quite local
in distribution. The existing collections include over 1700 specimens of
which 190 are only identified to family level. All the larger genera include
significant numbers of specimens not identified at the species level.

Many duplicates of the Riedelia collections from the former German
New Guinea were sent for study to Valeton in Bogor. Also, Herbarium
Bogoriense has many duplicates of the Riedelia types based on the
collections of Schlechter, which were described by Valeton. The problems
encountered by many specialists working on the New Guinean flora, the
destruction of critical types collections in Berlin, does not apply to this
genus. Recent ginger specimens are often poorly collected, many in fruit
but lacking flowers. The quality of the collections reflects the difficulties of
collecting in New Guinea.

A detailed taxonomic understanding, particularly of Riedelia and
Alpinia, will probably require the recollection (preferably from their type
localities) of most New Guinea species. Many of the collections are poor
(often due to insect damage in preservation) and have not been, probably
cannot be, identified at the level of genus and species.

The major requirements for collecting specimens of gingers are
outlined by Burtt and Smith (1976). Particular care should be made when
collecting the flowers of gingers. Spirit material and materials for DNA
studies could prove critical for an understanding of the larger genera in
New Guinea. Photographs of the plants and flower details will be critical.
The careful recording of ecological data is important. Future work should

102 Gard. Bull. Singapore 59 (1&2) 2007

also include living plant collections for growing in the botanic gardens at
Lae and Bogor, with duplicate plants sent to Singapore and Edinburgh for
additional planting, provided proper export permits are obtained.

No revision of the genera of the Zingiberaceae of New Guinea
should be attempted until a sustained effort has been made to recollect
adequate materials of the species from the type localities in order that the
many names can be properly applied. This applies not only to Riedelia but
to all the New Guinea genera of Zingiberaceae. Areas where the family is
not, or is under collected, should also be targeted for future expeditions.
Copies of the New Guinea database of the Zingiberaceae have been given to
National Herbarium in Lae, the Singapore Herbarium, and the Herbarium
of the Royal Botanic Gardens in Edinburgh.

Acknowledgements

I am very grateful to the Christensen Fund who provided a grant for studies
on the flora of New Guinea. Without this support the studies would not be
possible. I wish to express my sincere thanks to Dr. Jana Leong-Skornickova
(SING) who encouraged my interests in the Zingiberaceae while working
on the New Guinea database at the Singapore Botanic Gardens. Thanks are
due to the Directors of the following Herbaria for permission to examine
their collections: BO, BRI, CANB, LAE, SING. I also wish to thank to Dr.
Mark Newman (E) for sending me a copy of his unpublished paper on the
Zingiberaceae prepared for the ‘Ecology of Papua’, and also to thank the
two reviewers for their constructive comments.

References

Burtt, B.L. and R.M. Smith. 1972a. Key species in the taxonomic history
of Zingiberaceae. Notes from the Royal Botanic Garden, Edinburgh 31:
177-227.

Burtt, B.L. and R.M. Smith. 1972b. Notes on Malesian Zingiberaceae. Notes
from the Royal Botanic Garden, Edinburgh 31: 307-316.

Burtt, B.L. and R.M. Smith. 1976. Notes on the collection of Zingiberaceae.
Flora Malesiana Bulletin 29: 2599-2601.

Gilli, A. 1980 [publ.1983]. Beitrage zur Flora von Papue-New Guinea, III
Monocotyledones. Annalen des Naturhistorischen Museums in Wien, B,
84: 5-47.

New Guinea Database on Zingiberaceae 103

Johns, R.J., PJ. Edwards, T.M.A. Utteridge and H.C.F. Hopkins. 2006. A
guide to the alpine and subalpine flora of Mount Jaya. Kew Publishing,
Royal Botanic Gardens, Kew.

Larsen, K., JM. Lock, H. Maas and P.M. Maas. 1998. Zingiberaceae, pp.
474-495. In: Kubizki, K. (ed.): The Families and Genera of Vascular Plants,
Vol. IV. Springer-Verlag, Berlin, Heidelberg.

Loesener, T. 1930. Zingiberaceae. Die Natiirlichen Pflanzenfamilien (2™ ed.)
15a: 541-640, 654-693.

Newman, M.F. 2007. Zingiberaceae. pp. 473-476 In: A.J. Marshall and B.
Beehler (eds.). The Ecology of Papua. 5. Periplus (HK) Ltd Editions,
Singapore.

Newman, M., A. Lhuillier and A.D. Poulsen. 2004. Checklist of the
Zingiberaceae of Malesia. Blumea Supplement 16: 1-166.

Rickett, H.W. and FA. Stafleu. 1959. Nomina generica conservanda et
rejicenda spermatophytorum. Jaxon 8: 213-243.

Ridley, H.N. 1886. New species collected by Forbes. Journal of Botany 24:
356-358.

Ridley, H.N. 1916. Report of the Botany of the Wollaston Expedition to
Dutch New Guinea 1912-1913. Transactions of the Linnean Society,
Botany 9: 1-269.

Ridley, H.N. 1923. A contribution to our knowledge of the Flora of Papua
(British New Guinea). Proceedings of the Royal Society, Queensland 34:
19-21.

Riswan, S. and F.M. Setyowati. 1996. Ethnobotanical study of Zingiberaceae
in Indonesia, pp. 196-218. In: Wu, T.L., Wu, Q.G. & Chen, Z.Y. (eds.).
Proceedings of the 2" Symposium on the Family Zingiberaceae.
Zhongshan University Press.

Royen, P. van 1979. Zingiberaceae. In: The Alpine Flora of New Guinea 2:
860-866.

Schumann, K. 1899. Monographie der Zingiberaceae von Malaisien und
Papuasien. Botanische Jahrbiicher fiir Systematik, Pflanzengeschichte
und Pflanzengeographie 27: 259-350, t. 2-6.

104 Gard. Bull. Singapore 59 (1&2) 2007

Schumann, K. 1904. Zingiberaceae. In Engler, A. & Prantl (eds.), Das
Pflanzenreich IV, 46 (20): 1-458, figs. 1-52.

Smith, R.M. 1975. A preliminary review of the large bracteate species of
Alpinia. Notes from the Royal Botanic Garden, Edinburgh 34: 149-182.

Smith, R.M. 1990a. Alpinia (Zingiberaceae): a proposed new infrageneric
classification. Edinburgh Journal of Botany 47: 1-75.

Smith, R.M. 1990b. Psychanthus (K. Schum.) Ridley (Zingiberaceae): its
acceptance at generic level. Edinburgh Journal of Botany 47: 77-82.

Smith, R.M. 1990c. Pleuranthodium replaces the illegitimate name
Psychanthus (Zingiberaceae). Edinburgh Journal of Botany 48: 63-68.

Valeton, T. 1913a. Zingiberaceae. cones Bogoriensis 4(3): t. 373-375.

Valeton, T. 1913b. Zingiberaceae. Nova Guinea 8: 923-988, t. 162-179.

Valeton, T. 1914. Die Zingiberaceen Deutsch-Neu-Guineas. Botanische
Jahrbiicher fiir Systematik, Pflanzengeschichte und Pflanzengeographie
52: 41-100, figs. 1-11.

Valeton, T. 1917. Zingiberaceae. In: Gibbs, L.S., A Contribution to the

Phytogeography and Flora of the Arfak Mountains. Taylor & Francis,
London.

Gardens’ Bulletin Singapore 59 (1&2): 105-112. 2007 105

Morphology and Palynology of Amomum Roxb.
in Thailand

W. KAEWSRI' AND Y. PAISOOKSANTIVATANA ”

‘Department of Biotechnology, Faculty of Science, Mahidol University,
Kanchanaburi 71150, Thailand
Email: kawks@mahidol.ac.th
Department of Horticulture, Faculty of Agriculture, Kasetsart University,
Chatuchak, Bangkok 10900, Thailand
Email: agryyp@ku.ac.th

Abstract

Morphological characteristics and pollen morphology of Thai Amomum
Roxb. were studied in order to aid identification and classification. Thirty-
one species collected during our field expedition, only 13 species could be
identified to species, and 18 species will be proposed as new to science.
Investigation of both vegetative and reproductive organs reveals that
leaf, flower and fruit are useful for identification/separating the species
of Amomum. Pollen grains of 14 representatives were examined using a
scanning electron microscope (SEM) in order to reveal their morphology
and usefulness for infrageneric classification. Two types of exine sculpture,
psilate and echinate, were found. Classification by using pollen morphology
does not support grouping by the previous authors that emphasized fruit
shapes.

Introduction

Amomum Roxb. is one of the largest genera in the ginger family
(Zingiberaceae) with about 150-180 species. They are widely distributed in
Southeast Asia from the Himalayas to Northern Australia and extend into
the central Pacific (Kiew, 1982; Smith, 1985). Sirirugsa (2001) estimated
15-20 Amomum species in Thailand. They are generally evergreen herbs
inhabiting wet forests, especially in light gaps and at forest margins (Sakai
and Nagamasu, 1998). Many species of Amomum are used as medicine,
spice, condiment or a vegetable. Even though the plants from this genus
have been long utilized, the identification is still confusing because of the
absence of good specimens in many herbaria. Moreover, there are often
misidentified either infragenerically and intergenerically. These lead to

106 Gard. Bull. Singapore 59 (1&2) 2007

many changes in taxonomic status. For instance, many species of Amomum
were transferred to the other genera: Aframomum K. Schum., Elettariopsis
Baker, Alpinia Roxb., Etlingera Giseke and Hornstedtia Retz. Besides,
there are frequent changes in species identification. These problems need
an intensive study for clarification.

Thailand has a very complex biogeography due to its topography and
geographic position. Thus the southern Chinese flora reaches its southern
limits in Chiang Mai and Nan provinces, while the Malesian flora covers
the southern part of peninsular Thailand, the Burmese flora spills over the
western limestone mountains, and the central table mountains harbour
a rich endemic flora (Larsen, 2003). The diverse ecological habitats also
contribute to a rich diversity of plants including species of Amomum.

At present, the country forested area decreases rapidly due to
deforestation, urbanization and agricultural land expansion. Many plant
species in the forest include Amomum, are at risk of extinction. In order to
set conservation priorities for these potentially endangered species, the aim
of the present study is to examine the vegetative and reproductive parts, as
well as pollen morphology, useful for classification.

Materials and Methods

Indigenous species of Amomum were collected from all parts of Thailand
during April 2003-June 2005 (Table. 1). Inflorescences, infructescences,
vegetative parts and habitats, were photographed, and field notes were made.
Flowers and fruits were preserved in 70% ethanol. The dried vegetative
part and inflorescences were deposited at the herbaria BK (Department
of Agriculture, Bangkok) and BKF (Royal Forest Department, Bangkok),
Thailand. Living specimens are cultivated at the Department of Horticulture,
Kasetsart University. Morphological characters were examined either from
dry, spirit collections or living plants. Anthers of 14 species of Thai Amomum
were taken from live plants at mature stage and stored in 70% ethanol.
Pollen grains were collected and kept also in 70% ethanol. The samples
were passed through an ethanol dehydration starting with 10-15 min in 90%
ethanol with three subsequent changes in absolute alcohol at 10 min each.
The Critical Point Dryer (BALZERS LINION CPD-020) was used to dry
the samples. The pollen were then mounted on SEM stub using double-
sided sticky tape and sputter-coated. Photographs were taken with a JSM
Jeol 5410LV scanning electron microscope.

Morphology and Palynology of Amomum Roxb. in Thailand

107

Table 1. List of Amomum species collected in Thailand during April 2003-June 2005.

No. Botanical Name

BO CEN Se, eee

Amomum aculeatum Roxb.
Amomum biflorum Jack
Amomum dealbatum Roxb.
Amomum hastilabium Rid.
Amomum koenigii J.F. Gmel.
Amomum micranthum Ridl.
Amomum pierreanum Gagnep.

Amomum repoeense Pierre

ex Gagnep.

Amomum rivale Ridl.

Amomum siamense Craib
Amomum testaceum Ridl.
Amomum uliginosum Konig

Amomum cf. villosum

Amomum sp.1
Amomum sp.2
Amomum sp.3
Amomum sp.4
Amomum sp.5
Amomum sp.6
Amomum sp.7
Amomum sp.8
Amomum sp.9

Amomum sp.10

Amomum sp.11
Amomum sp.12
Amomum sp.13
Amomum sp.14
Amomum sp.15
Amomum sp.16
Amomum sp.17

Amomum sp.18

Collector No.

Kaewsri 2, 6, 20, 65, 74

Kaewsri 52, 58, 66
Kaewsri 110
Kaewsri 37

Kaewsri 03, 29, 136
Kaewsri 63, 84
Kaewsri 122
Kaewsri 64, 103, 121

Kaewsri 04, 23, 142
Kaewsri 14, 116, 123
Kaewsri 15, 16, 96
Kaewsri 30, 32, 33, 92
Kaewsri 12, 13
Kaewsri 01, 93
Kaewsri 10, 139
Kaewsri 19, 75
Kaewsri 22

Kaewsri 24, 147

Kaewsri 25, 88, 89, 113

Kaewsri 27
Kaewsri 35

Kaewsri 38

Kaewsri 50, 54, 104, 105,

107

Kaewsri 68, 79
Kaewsri 70

Kaewsri 81

Kaewsri 93, 94
Kaewsri 97, 108
Kaewsri 111, 114, 137
Kaewsri 134, 135
Kaewsri 151

Locality
Kanchanaburi
Chanthaburi
Chiang Mai
Ranong
Kanchanaburi
Chanthabur1
Nakhon Nayok
Chanthaburi

Kanchanaburi
Tak

Tak (Cultivated)
Nakhon Nayok
Tak
Kanchanaburi
Kanchanaburi
Prachuap Khiri Khan
Kanchanaburi
Kanchanaburi
Kanchanaburi
Uthai Thani
Ranong

Ranong

Sakon Nakhon

Chanthaburi
Chumphon
Ranong

Tak

Chiang Mai
Chiang Mai
Nan

Kanchanaburi

108 Gard. Bull. Singapore 59 (1&2) 2007

Results and Discussion

1. Morphological characters of vegetative and reproductive parts

Thirty-one species of Amomum collected from all over Thailand including
13 identified species and 18 unidentified species (Table 1) were examined.
The sheath surface was either reticulate (Fig. 1A) or triate (Fig. 1B), and
the ligule, either bilobed (Fig. 1C) or entire (Fig. 1D). These characters have
only limited application in species determination whereas reproductive parts
are of more useful to aid identification. The latter includes the differences
in shape of the stigma (Fig.1E-G), lateral staminodes (Fig.1H-J), labellum
(Fig.1K-N), anther crest (Fig.1O-S), and fruit (Fig.1T-W).

Figure 1. Morphological characteristics of some organs of the genus Amomum Roxb.

Leaf sheath: A. reticulate; B. striate. Ligule: C. bilobed; D. rounded. Stigma: E. cup shaped;
F. ampullate; G. clavate. Lateral staminode: H. subulate; I. fin-shaped; J. linear. Labellum:
K. ovate; L. deltoid; M. orbiculate and hooded; N. flabellate. Anther crest: O. reniform:; P.
auriculate; Q. trilobed or horn-shaped; R. truncate; S. rounded. X-section of fruit: T. smooth;
U. ridged; V. spiny; W. wing.

Morphology and Palynology of Amomum Roxb. in Thailand 109

Due to the closely resemblance in their floral morphology, it is
recommended that the fruit types be examined. Three major types of fruits
(smooth, hairy and winged) are found among Thai species of Amomum.
Some variation in the degree of hairiness and smoothness of the fruit are
also observed. The fruit shape is highly variable and can be globose, ellipsoid,
obovoid, or obconical (Fig. 2A-M).

Figure 2. Fruit shapes of Amomum: A. Amomum aculeatum; B. A. biflorum; C. A. dealbatum;
D. A. rivale; E. A. siamense; F. A. testaceum; G. A. uliginosum;H. Amomum sp.3; I. Amomum

sp.4; J. Amomum sp.6; K. Amomum sp.11; L. Amomum sp.12;M. Amomumi sp.15.

2. Palynological study

The SEM results revealed that the pollen grains of Thai Amomum are
spherical to sub-spherical, (except for some grains of A. rivale Ridl. that
are ovoid), 30-70 um in diameter and the intine layer is 1-7 um thick. These
general results agree with Mangaly and Nayar (1990) which reported that
the pollen shape of Amomum hypoleucum Thwaites and A. pterocarpum
Thwaites are sub-spheroidal to ovoid and spheroidal, the diameter being
30-90 and 35-75 um, respectively and the intine layer 1.25-2.5 um thick in
Amomum hypoleucum.

Our results revealed that the pollen can be divided into two
groups either by intine thickness (<4 um; =4 um), and by exine sculpture
(echinate or psilate; Table 2, Fig. 3). The results do not coincide with the
classification proposed by previous authors, in particular, Schumann (1904)
who subdivided the genus (section Euamomum) into two series, namely,
1. ser. Lobulatae (anther crest with two or three lobes) and 2. ser. Integrae
(anther crest margin with entire lobe). Our study reveals that most pollen
grains are echinate in sculpture, which was consistent in both series and are
of less use in classification.

110 Gard. Bull. Singapore 59 (1&2) 2007

As stated above, the results from palynological study are not useful
for classification of Thai species of Amomum. Most data from pollen
shapes and size characteristics are apparently homoplasious although exine
sculpture can divide the species into two groups: echinate and psilate. The
echinate group consists of A. aculeatum, A. biflorum, A. dealbatum, A.
siamense, A. uliginosum, Amomum sp. 3, Amomum sp. 4, Amomum sp. 6,
Amomum sp. 11, Amomum sp. 12, Amomum sp. 15, Amomum sp. 16. The
psilate group includes only two species: A. rivale and A. testaceum.

Table 2. Pollen morphology of Thai Amomumi species.

Shape and size Intine
Species (aa) knees Exine sculpture
1. A. aculeatum Spherical, 3 um Echinate; spine uniformly
46-60 um. distributed, sharp apex, having
collared base, 1.5-3 um tall, 2-3 um
thick at base.
2. A. biflorum Spherical, 1-2 um Echinate; spine uniformly
35-46 um. distributed, usually sharp apex,
having collared base, 2.5-3 um tall,
3-3.5 um thick at base.
3. A. dealbatum Spherical, 3 um Echinate; spine uniformly
52-58 um. distributed, blunt and interrupted
by sharp apex, 3-3.5 um tall, 3 um
thick at base.
4. A. rivale Subspherical 1 um Psilate; exine 0.5 um thick
or ovoid,
ca. 60 um.
5. A. siamense Spherical, 5 um Echinate; spine uniformly
43-53 um. distributed, sharp apex, 2.5-3 um
tall, 3 um thick at base, surface
reticulate.
6. A. testaceum Spherical to 2 um Psilate; exine 1 um thick.
subspherical,
60 um.
7. A. uliginosum Spherical, 1-2 um Echinate; spine irregularly
30-35 um. distributed, blunt interupted by
sharp apex, having collared base, 1
um tall, 2 um thick at base.
8. Amomum sp.3 Spherical, 4-5 um Echinate; spine uniformly
63-70 um. distributed, distributed, sharp and

interrupted by blunt apex, 4 um
tall, 6 um thick at base.

Morphology and Palynology of Amomum Roxb. in Thailand

9. Amomum sp.4

10. Amomumi sp.6

11. Amomum sp.11

12. Amomum sp.12

13. Amomum sp.15

14. Amomum sp.16

Spherical or
subspherical,

50-58 um.

Spherical to
subsperical,

45-50 um.

Spherical,

56-60 um.

Spherical,

60-64 um.

Spherical,
50-60 um.

Spherical,
60-63 um.

1 um

4-5 um

4-7 um

1 um

2-3 um

ISkU X5686

ttt

Echinate; spine uniformly
distributed, sharp or blunt apex,
having collared base, ca 2 um tall,
3 um thick at base.

Echinate; spine uniformly
distributed, blunt and interrupted
by sharp apex, 2-2.5 um tall,2-3
um thick at base.

Echinate; spine uniformly
distributed, sharp apex, having
collared base, 3 um tall, 3 um thick
at base.

Echinate; spine uniformly
distributed, sharp apex, 3-4 um
tall, 2-2.3 um thick at base.

Echinate; spine uniformly
distributed, sharp apex, 4 um tall,
3.5 um thick at base.

Echinate; spine uniformly
distributed, sharp apex, 4 um tall,
4 um thick at base.

S@um 261132

Figure 3. Pollen characteristics of Amomum. Psilate type: A. A. rivale; B. A. testaceum.
Echinate type: C. A. aculeatum. Echinate-reticulate type: D. A. siamense.

112 Gard. Bull. Singapore 59 (1&2) 2007

Conclusions

1. Morphology of reproductive parts is more useful in aiding precise
species identification of Amomum, especially the fruit shape.

2. The pollen grains of Amomumare spherical to subspherical,inaperturate,
and the exine sculpture is either echinate or psilate. Pollen characteristics
agree with the previous reports but do not correspond with previous
classification based on morphological characteristics. Therefore,
the pollen morphology is less useful for subgeneric classification of
Amomum.

References

Kiew, K. Y. 1982. The genus Elettariopsis (Zingiberaceae) in Malaya.
Notes from the Royal Botanical Garden Edinburgh 42: 295-314.

Larsen, K. 2003. The Zingiberaceae in Flora of Thailand, pp. 1-5. In: P.
Chantaranothai, K. Larsen, P. Sirirugsa and D.Simpson (eds.). Proceedings
of the 3rd Symposium on the Family Zingiberaceae. Applied Taxonomic
Research Center, Department of Biology, Faculty of Science, Khon Kaen
University.

Mangaly, J. K.and J. Nayar. 1990. Palynology of south Indian Zingiberaceae.
Botanical Journal of Linnaean Society 103: 351-366.

Sakai, S. and H. Nagamasu. 1998. Systematic Studies of Bornean
Zingiberaceae I. Amomum in Lambir Hills, Sarawak. Edinburgh Journal
of Botany 55: 45-64.

Schumann, K.M. 1904. Zingiberaceae. In: A. Engler (ed.), Das Pflanzenreich
IV, 46: 1-458.

Sirirugsa, P. 2001. Zingiberaceae of Thailand, pp. 63-77. In: V. Baimai and
R. Kumhom. BRT Research Reports 2001. Biodiversity Research and
Training Program. Jirawat Express Co., Ltd., Bangkok. (in Tha1)

Smith, R.M. 1985. A review of Bornean Zingiberaceae:1(Alpineae). Notes
from the Royal Botanical Garden Edinburgh 42: 295-314.

Gardens’ Bulletin Singapore 59 (1&2): 113-128. 2007 113

An analysis of generic circumscriptions in tribe Alpinieae
(Alpiniodeae: Zingiberaceae)

‘W.J. KRESS ',M.F NEWMAN”, A.D. POULSEN”
AND C. SPECHT °*

‘ Department of Botany, MRC-166, United States National Herbarium, National
Museum of Natural History, Smithsonian Institution, P.O. Box 37012, Washington,
D.C. 20013-7012 USA
: Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, Scotland, UK
* Department of Plant and Microbial Biology, University of California, Berkeley,
431 Koshland Hall, MC 3102, Berkeley, CA 94720 USA

Abstract

Recent investigations based on molecular phylogenies have resulted in
new insights into the evolutionary relationships and classification of the
Zingiberaceae and various genera within the family, e.g., Globba, Hedychium,
Roscoea, Etlingera, Alpinia, and Amomum. At the same time taxonomic
boundaries of many traditionally recognized genera have been challenged,
e.g., Curcuma, Boesenbergia, Caulokaempferia, Alpinia, and Amomum.
Within the subfamily Alpinioideae the results of our analyses will require
the recircumscription of many of the genera included in the tribe Alpinieae.
These phylogenetic results are based on a supermatrix analysis of ITS and
matK sequence data and are discussed in the context of complementary
morphological features and geographic distributions. Seventeen clades
are recognized at the generic level although some remain tentative and in
need of additional analysis before final taxonomic circumscriptions can be
made. A revised classification will require that many species be placed in
new or different genera, which will greatly facilitate identification and our
understanding of morphological evolution in the family as well as species
and genera therein.

Introduction

During the last few years molecular data have been routinely used in
determining generic boundaries and evolutionary relationships of the
genera in the ginger family, the Zingiberaceae (e.g., Harris et al., 2000;
Ngamriabsakul et al., 2000; Rangsiruji et al.,2000a, b; Searle and Hedderson,

114 Gard. Bull. Singapore 59 (1&2) 2007

2000; Wood et al., 2000; Kress et al., 2002; Pedersen, 2004; Williams et al.,
2004; Xia et al.,2004). The study by Kress et al. (2002) is the most thorough
paper to date addressing relationships among genera in the Zingiberaceae.
In that study, sequence data from both the Internal Transcribed Spacer
(ITS) and matK regions were used to establish well-resolved phylogenetic
relationships among the genera, and a new classification of the Zingiberaceae
was proposed that recognized four subfamilies and four tribes. Kress et
al. (2002) also demonstrated that a number of the larger genera in the
family (Amomum, Alpinia, Boesenbergia, and Curcuma) may be para- or
polyphyletic and suggested that more extensive sampling was necessary
for these taxa. Subsequent studies have been carried out in some of them
(Alpinia: Kress et al., 2005; Amomumi: Xia et al., 2004).

With respect to the tribe Alpinieae, the results of investigations by
Rangsiruji et al., (2000a, b), Kress et al., (2002, 2005), and Xia et al. (2004)
are most pertinent. In the first study, in which 47 species of Alpinia and a
small number of outgroup taxa were sampled, the authors demonstrated
significant statistical support for several monophyletic groups of species of
Alpinia, but suggested that the genus itself may not be monophyletic. In
a broader analysis of genera of Alpinioideae, Kress et al. (2002) identified
four separate groups of alpinias (Alpinia I-IV) for the 11 Alpinia species
sampled (Fig. 1). These four groups did not form a monophyletic assemblage,
were scattered throughout the tribe, and corresponded to at least some of
the clades recognized in the molecular analyses of Rangsiruji et al. (2000b).
Further sampling in the tribe was also conducted by Xia et al. (2004) in
their analysis of the generic boundaries of Amomum. They identified at
least three major non-monophyletic groups of species within the current
circumscription of Amomum. Within Alpineae, the phylogenetic position
of the presumed extinct Leptosolena and a second species of the formerly
monotypic Vanoverberghia were determined by Funakoshi et al. (2005)
based on molecular sequence data, providing more information on generic
relationships within the tribe.

Kress et al. (2005) conducted the most exhaustive phylogenetic
analysis of tribe Alpinieae to date sampling 99 species in the Alpinioideae
with an emphasis on the genus Alpinia. Their results (Fig. 2) demonstrated
six polyphyletic clades of Alpinia and at least two clades of Amomum
while resolving the evolutionary relationships among a number of genera
in the Alpinieae with a slightly different topology than earlier analyses
had indicated (Kress et al., 2002). Although their analyses confirmed the
division of both Alpinia and Amomum into numerous polyphyletic groups,
the authors were reluctant to propose a new classification until a number of
issues, especially taxon sampling, were resolved.

An analysis of generic circumscriptions in tribe Alpinieae 115

Aframomum
Reneaimia
Amomum I
Efettariopsis
Paramomum
Alpinia I
Plagiostachys

Alpinieae Alpinioideae
Vanoverberghia

Etlingera

Hornstedtia

Amomum II

Pleuranthodium ; 5
Riedelia Riedelieae

Siamanthus
Siliquaamomum Incertae sedis

Figure 1. Early results on the phylogenetic relationships among the genera of the subfamily
Alpinioideae of the Zingiberaceae based on a parsimony analysis of ITS and matK sequence
data for 45 species in the subfamily (from Kress et al., 2002). Note the four polyphyletic
clades of Alpinia and two clades of Amomum.

Aframomum
Reneaimia
Aipinia Fax clade

Buse oleioat
Elettariopsis
ig thecarieetaes

Aipinia Galanga clade

‘Alpinia Carolinensis clade | Alpinieae
‘ipinia Zerumbet clade |
Alpinia Eubractea clade Alpinicideae
Etlingera
Hornstedtia
Amomum II
‘ipinia Raffiesiana clade
as Neeaeegair

Pleuranthodium Riedelieae

Riedelia
iamanthus

Figure 2. Most recent results on the phylogeny of the Alpinioideae resulting from further
analysis of the combined ITS and matK sequence data for 99 species in the subfamily (from
Kress et al. 2005). Note the six clades of Alpinia. The Alpinia Zerumbet clade includes the
genus Plagiostachys; the Alpinia Eubractea clade includes the genera Vanoverberghia and
Leptosolena.

116 Gard. Bull. Singapore 59 (1&2) 2007

The goals of our current analyses are 1) to combine molecular
sequence data from previously published analyses with new original
sequences to build a “supermatrix” for Alpinieae; 2) to identify major clades
defined by molecular data and assess their correspondence to existing
generic boundaries; and 3) to provide new circumscriptions of genera and
apply new names if necessary. Goals one and two are addressed in this
paper. The results of these analyses have established the major lineages
within the tribe, have confirmed the validity of previously recognized genera,
and have identified clades which will require new generic circunscriptions.
Although our sampling is extensive, we will be adding several key taxa to a
final analysis to be published in the near future. At that time we will address
goal three and provide a new revised classification of the Alpinieae that will
recognize more finely circumscribed genera.

Materials and Methods

Taxon and Character Sampling

Taxon sampling was designed to include the full diversity (taxonomic,
morphologic, and biogeographic) of the Alpinioideae with a focus on
phyletic and biogeographic diversity in Alpinia and Amomum. A total of
230 taxa are used in the analysis, including 23 outgroup taxa representing the
Zingiberoideae, Tamijiodeae and Siphonochiloideae. Previously published
sequence data from our own work as well as additional data downloaded
from GenBank were combined with new original sequences in the current
analysis. Two independent gene regions were sampled for this analysis: the
ITS (internal transcribed spacer) region of nuclear ribosomal DNA (White
et al., 1990) and the intron of the chloroplast transfer RNA gene for lysine
trnK, including the maturase (matK) coding region and S’and 3’ flanking
introns (Johnson and Soltis 1994; Mohr et al. 1993). ITS was obtained for
all 230 taxa included in this analysis, while the entire trnK region is missing
from 84 taxa.

DNA Isolation and Manipulation

Whole genomic DNA was extracted from plants with the Plant DNAeasy
kit protocol (Qiagen). DNA fragments were amplified and sequenced
for each of the gene regions using the primers and protocols previous
published for Zingiberales (Kress et al., 2002, 2005; Specht, 2006; Specht er
al.,2001). Sequencing was carried out on an ABI 3700 automated sequencer
equipped with ABI PRISM ~ sequencing analysis software. Sequences
were analyzed and edited using Sequence Navigator (Applied Biosystems)
and GeneJockey (Taylor, 1994) or Se-Al (Rambaut, 1996). Alignments

An analysis of generic circumscriptions in tribe Alpinieae aTy

across taxa were performed using CLUSTAL X (Thompson et al. 1994) as a
Multiple Alignment option in GeneJockey with both fixed and floating gap
penalties set to 10. All manual alignment adjustments follow the criteria of
Zurawksi and Clegg (1987) in which gaps are considered as characters and
the number of evolutionary events (insertions or deletions) is minimized.
All characters are treated as unordered and gapped regions are treated as
missing, however insertion-deletion events (indels) are coded as additional
individual binary characters using the simple indel coding method (Simmons
and Ochoterena 2000). A total of 25 and 17 gaps were coded from within
the trnK and ITS regions, respectively.

Phylogenetic Analysis

The combined ITS and trnK/matK phylogenetic analyses were performed
using the parsimony optimality criterion,in each case considering all positions
of equal weight for evaluating phylogenetic relationships. Analyses were
conducted with PAUP*4.0b4a (Swofford, 2001) for a total of 3820 aligned
characters with 28% of the matrix scored as missing as the result of either
inability to acquire sequence data for certain taxa or the presence of gap
characters. For all parsimony analyses, heuristic searches were performed
with TBR as the branch-swapping algorithm; starting trees were obtained
using stepwise random addition with 100 replicates and one tree held at
each step. Jackknife support values were calculated with 37% deletion and
the “emulate Jac resampling” option selected (Farris et al., 1996).

Results and Discussion

The first goal of our analysis was addressed by the parsimony consensus
tree of the combined ITS and trnK/matK sequence data. The consensus
tree represents 9,624 equally parsimonious trees with a length of 4,277
steps, including 32 coded gaps (Figs. 3, 4). A total of 920 characters were
parsimony informative (2433 were constant, 467 were uninformative). The
majority of primary clades defining recognized or tentative genera, including
Etlingera, Renealmia, Aframomum, the Alpinia rafflesiana clade, the Alpinia
zerumbet clade, the Alpinia carolinenesis clade, the Alpinia eubractea clade,
the Alpinia galanga clade, the Alpinia fax clade, and the Amomumi tsao-
ko clade, are strongly supported with jackknife values ranging between
99-100%. Monotypic genera or genera with only a single species sampled
in our analysis, including Siliquamomum, Geostachys, and Geocharis, are
clearly differentiated from their sister taxa. The Amomum villosum clade,
the Amomum maximum clade and the Elettariopsis clade have slightly lower
jackknife values (76-89%) while the seven species of Hornstedtia sampled

118 Gard. Bull. Singapore 59 (1 &2) 2007

in our analysis form a grade with Etlingera. The relationships of only a few
of the major lineages are not fully resolved, such as the placement of the
Siliquamomum-Alpinia rafflesiana clade, and the polytomy formed by the
Geostachys - Amomumi tsao-ko clade, the Alpinia zerumbet clade, and the
Alpinia carolinenesis clade.

The second goal of our analyses, to assess the correspondence of
the major clades defined by the molecular data to existing generic concepts,
has also been accomplished. The 17 clades recognized here (Fig. 4) can be
classified into three categories: 1) well-supported lineages that correspond to
formerly recognized genera whose taxonomic names should be maintained,
2) well-supported lineages in need of new (or previously used) taxonomic
names, and 3) problematic lineages in need of additional data prior to
making final taxonomic decisions.

Figure 3. The strict consensus supertree of 9,624 equally parsimonious trees of the
Alpinioideae (with an emphasis on tribe Alpinieae) in the analysis of combined ITS and
trnK/matK sequence data for 207 species in the subfamily (length = 4,277 steps) showing
bootstrap values from the parsimony analysis. The seventeen major clades in Alpinieae are
indicated with small letters (a through q) and variously color shading. For discussion of each
clade see text.

An analysis of generic circumscriptions in tribe Alpinieae 119

Aframomum
‘Renealmia
Alpinia fax clade
Amomum maximum clade}
Elettariopsis clade |
Alpinia galanga clade
| Etlingera
Hornstedtia grade

Alpinia eubractea clade | Alpinieae

d
Amomum viflosum clade |

Alpinioideae
Geocharis

Alpinia carolinensis clade
Alpini zerumbet clade
Amomum tsao-ko clade
Geostachys

Alpinia rafflesiana clade |
Siliquamomum |
Burbidgea ch
Pleuranthodium
Riedelia

Siamanthus

Riedelieae

Figure 4. Condensed tree of the Alpinioideae resulting from the analysis of the combined
ITS and trnK/matK sequence data (see Fig. 3) in which the major clades/grades have been
collapsed into single branches for clarity.

(1) Well-supported lineages that correspond to formerly recognized genera
whose taxonomic names should be maintained.

Aframomum. As earlier demonstrated by Harris et al. (2000), this African
genus is strongly supported as monophyletic by molecular data. The flask-
shaped fruit is a distinct synapomorphy of the genus. The basal inflorescence
radical to the leafy shoot is shared with its sister taxon Renealmia from which
it is distinquished by the presence of scale-like trichomes on the vegetative
structures in Aframomum.

Renealmia. This genus is one of the few genera in the order Zingiberales
with an amphi-Atlantic distribution with species in the tropical forests of
the Americas and Africa. The clade is well-supported as monophyletic, and
although it shares the distinctive basal inflorescence with Aframomum, it is

120 Gard. Bull. Singapore 59 (1&2) 2007

distinguished by stellate, rather than scale-like, trichomes. It should be noted
that at least a few species on both sides of the Atlantic have inflorescence
terminal on the leafy shoots (e.g., the neotropical R. cernua (Sw. ex. Roem.
& Schult.) J. F Macbr., R. helenae Maas, and R. pyramidalis (Lam.) Maas,
and the African R. battenbergiana Cummins).

Alpinia galanga clade. The type species of the large conglomerate genus
Alpinia is A. galangal (L.) Willd., which is placed in this small clade in the
molecular analyses. For this reason, the generic name A/pinia is best applied
to this group of four species. Both the placement of this clade in relationship
to other genera and the monophyly of the four species have strong jackknife
support (100% ). Branched inflorescence with small flowers, open bracteoles,
a clawed labellum, and thin-walled fruits are characteristic of the species in
the A. galanga clade. Members of this clade are distributed primarily in
continental Asia with the wide distribution of A. galanga most likely due to
its important culinary use by local peoples.

Etlingera. The often large involucre of sterile inflorescence bracts and
the fusion of the corolla tube to the labellum and single stamen filament,
forming a staminal tube beyond the insertion of the corolla lobes, are
characteristic of this monophyletic genus. Species are spread throughout
the wet lowland tropics of Southeast Asia. The genus Eftlingera 1s closely
related and apparently paraphyletic with Hornstedtia.

Geocharis. Although only one of the six species of Geocharis was sampled in
our molecular analysis, this genus appears to be distinctive in the tribe being
differentiated by the radical, sometimes lax, inflorescence and the stem
venation marked by prominent white hairs between major veins. However,
increased sampling of species of this genus and other taxa placed in the genus
Amomum are needed to determine the monophyly of Geocharis. Species
are found in the Philippines, Peninsular Malaysia, Sumatra, and Borneo.

Geostachys. As in Geocharis, only one of the 20-25 species of this genus
was included in our analysis. The stilt roots, lax inflorescence, non-imbricate
bracts, and two or more flowers per cincinnus are characteristic of species
of Geostachys and suggest that the genus is monophyletic. Many endemic
species are found in this genus distributed in peninsular Malaysia, Sumatra,
northwestern Borneo, and Thailand to Cambodia.

Siliguamomum. With only a single species found in northern Vietnam and
bordering regions of tropical China, this genus has unique cylindrical torulose
fruits resembling siliques. The phylogenetic placement of Siliquamomum has

An analysis of generic circumscriptions in tribe Alpinieae 121

been problematic and often unresolved within the Alpinioideae (see Kress et
al., 2002, 2005). Our current analysis allies it to the Alpinia rafflesiana clade
although this placement may change in future analysis. It is best maintained
as a distinctive monotypic genus in the subfamily.

(2) Well-supported lineages in need of new (or previously used) taxonomic
names.

Alpinia fax clade. The three species placed in this clade (only two sampled
in the molecular study; the third species, A. rufescens (Thw.) K. Schum., is
only known from the type specimen) form a well-supported monophyletic
group characterized by a radical capitate inflorescence often borne on a
long leafless peduncle with conspicuous sterile bracts (Sabu, 2006). This
clade is distributed in Sri Lanka and southern India and is sister to the clade
containing the genera Renealmia and Aframomum (see above). Together
with these two genera the A. fax clade constitutes a strongly supported
monophyletic lineage stretching from tropical America through Africa to
south Asia, which may represent both vicariant and long-distance dispersal
events. All three species of this clade were originally described in the genus
Elettaria, which has not been included in the present analysis. A new generic
name is required for this clade.

Alpinia eubractea clade. This clade is made up of species found primarily
in the Pacific Ocean, including the Philippines, Oceania, and Australia, and
includes taxa earlier described in three genera (Alpinia, Leptosolena, and
Vanoverberghia). The A. eubractea clade is strongly supported (bootstrap =
100%) in the molecular analysis, but the morphological apomorphies of this
group of species are not immediately obvious. This clade will require a new
generic name after additional taxa are added to the phylogenetic analysis,
especially species of A/pinia from New Guinea.

Amomum villosum clade. Although it may be premature to recognize
segregate genera for species that were earlier described in the genus
Amomum, the results of our analyses as well as earlier investigations by Xia
et al. (2004) support at least three distinct lineages of taxa formerly placed
in Amomum. The A. villosum clade is characterized by echinate fruits and
a trilobed anther appendage. Species in this clade are distributed primarily
in Indochina, peninsular Malaysia, and Borneo. Additional species samples
throughout this distribution will provide a more complete picture of the
taxonomic breadth of this clade.

Alpinia carolinensis clade. Species in this clade tend to be plants large in

122 Gard. Bull. Singapore 59 (1&2) 2007

stature, with a caducous primary inflorescence bract, and a narrow fleshy
labellum adpressed to the stamen. Members of the A. carolinensis clade are
concentrated in Sulawesi and generally east of Wallace’s Line in the Pacific
Ocean. Additional species from Sulawesi and New Guinea should be added
to the molecular analysis in anticipation of applying a new generic name to
this clade.

Alpinia_zerumbet clade. The great bulk of species named in the genus
Alpinia are found in this well-supported clade with a broad geographic
distribution in tropical Asia. The absence of a primary inflorescence bract
and the presence of short one-three flowered cincinni characterize most of
the species included in the A. zerumbet clade. At least four subclades with
strong (= 100%) jackknife support are apparent within the A. zerumbet
clade, including the A. aquatica subclade, the A. nutans subclade, the A.
calcarata subclade, and the Plagiostachys subclade. It may be appropriate
to recognize each of these well-supported subclades at the subgeneric level.
However, if subgenera are to be established, at this time we advocate for the
purpose of simplicity the recognition of only two subgenera corresponding
to the A. zerumbet subclade and the Plagiostachys subclade as circumscribed
in Fig.3.

Amomum tsao-ko clade. The species included in this clade are primarily
Chinese in distribution. They are characterized by leaves with pleasant and
distinctive aromatic oils, a bi- or tri-lobed anther appendage, and smooth
fruits. In the molecular analysis this clade of Amomum is only moderated
supported as sister to Geostachys, which is morphologically distrinct. As
more species of Amomum are added to the overall analsyses, additional
species may be included in the A. tsao-ko clade.

Alpinia rafflesiana clade. Only two of the over 200 species in tribe Alpinieae
that wesampled are contained in this well-supported clade found in peninsular
Malaysia and southern Thailand. The two species are characterized by a
broadly spread labellum and/or drooping inflorescence. It is possible that
after additional sampling other species will be included in this clade, such
as A. capitellata Jack from Borneo, but the molecular distinctiveness of the
lineage suggests that a separate generic name is warranted. Our analysis has
placed the A. rafflesiana clade sister to the unique monotypic Siliquamomum,
which should be maintained as a separate genus (see above).

(3) Problematic lineages in need of additional data prior to making final
taxonomic decisions.

An analysis of generic circumscriptions in tribe Alpinieae 123

Amomum maximum clade. Species possessing an entire anther appendage,
orange and yellow labellum, and winged fruits are contained within this clade,
which was also recognized by Xia et al. (2004) in their molecular analysis of
Amomum. Most of the taxa are distributed in China, India, and Australia. In
our current sample of taxa the molecular data only moderately support the
monophyly of the A. maximum clade. However, jackknife support is strong
(100%) for the node joining the A. maximum clade with the Elettariopsis
clade (see below). Although we have not yet obtained sequence data for
A. subulatum Roxb., the type of the genus Amomum with winged fruits, we
expect this species to be included in one of these clades and not in the A. tsao-
ko clade or A. villosum clade. More sampling of species of Amomum with
winged fruits, including the type species, is needed before a final decision
can be made on taxonomic alignments.

Elettariopsis clade. This clade contains species of Elettariopsis, Amomum,
and the monotypic Paramomum and, similar to the A. maximum clade, it 1s
only moderately supported by our molecular data (jk = 89%). Elettariopsis
has long been recognized as a genus because of the distinctive two-to-three-
leaved shoots with characteristic aromatic oils, an elongated underground
inflorescence with subterranean fruits, and a trilobed anther crest. Some of
these features are also shared with the species of Amomum and Paramomum
contained in the same clade. If sampling additional taxa does not provide
better support for this clade, it may be justified to recognize the Elettariopsis
clade together with the A. maximum clade as a single genus.

Hornstedtia_ grade. The characteristic stilt roots and elongated corolla
tubes are found in most species of the widespread tropical Asian genus
Hornstedtia. Although the close relationship between this genus and
Etlingera has been recognized for over a half century (Holttum, 1950; Smith,
1985), the monophyly of Hornstedtia had not been challenged until recently.
Pedersen (2004) in her analysis of Etlingera demonstrated that the species
of Hornstedtia that she sampled formed a paraphyletic group. Our analyses
with broader sampling confirm her result. At this point we suggest that
Hornstedtia and Etlingera remain as separate genera until further analyses
clarify the issue.

Several interesting genera in the Alpinieae were not sampled in our
molecular analyses because of the unavailability of sufficient tissue samples
or problems encountered in generating DNA sequences. Cyphostigma, a
monotypic genus endemic to Sri Lanka and distinctive in its lax inflorescence,
resupinate flowers, and large petaloid anther crest, may be related to
Elettariopsis or the Amomum maximum clade because of similarities

124 Gard. Bull. Singapore 59 (1&2) 2007

in inflorescence and floral features (Holttum, 1950). Elettaria, which has
a broad distribution in India, peninsular Malaysia, Java, and Borneo and
contains the economically important spice cardamon (E. cardamomum (L.)
Maton), was also not sampled in our analysis. Its position in the Alpinieae
is as yet unclear.

Conclusions

As DNA sequence data for additional taxa have been added to the
phylogenetic analysis of the Alpinioideae, a more detailed and refined
understanding of evolutionary relationships and generic boundaries in the
subfamily has been obtained (Figs. 1-4). As outlined above, our investigations
are centered on three goals, two of which are addressed in this paper. We
will address the third goal (to provide new circumscriptions of genera and
apply new names where necessary) in a future publication that will include
a more complete sampling of critical taxa necessary to make final decisions
on meaningful taxonomic units in the Alpinioideae and especially the tribe
Alpinieae.

Acknowledgements

We wish to thank Ray Baker, Mike Bordelon, Mark Collins, Heather Driscoll,
David Harris, Qing-Jun Li, and Ida Lopez for assistance in preparing this
manuscript. Funding was provided by the Smithsonian Institution.

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Gardens’ Bulletin Singapore 59 (1 &2): 129-138. 2007 129

Materials for a Taxonomic Revision of Geostachys
(Baker) Ridl. (Zingiberaceae) in Peninsular Malaysia

K.H. LAU , C.K. LIM’ AND K. MAT-SALLEH °

"Tropical Forest Biodiversity Centre,
Biodiversity and Environment Division, Forest Research Institute Malaysia,
52109 Kepong, Selangor, Malaysia.
"215 Macalister Road, 10450 Penang, Malaysia.
“School of Environmental and Natural Resource Sciences,
Faculty of Science and Technology, Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor, Malaysia.

Abstract

Materials for a taxonomic revision of the Geostachys (Baker) Ridl. in
Peninsular Malaysia, resulting from recent fieldwork are presented, with
notes on the threat assessment of extant species. Twelve of the 13 previously
known species were studied in situ, and two newly described species have also
been found (Geostachys belumensis C.K. Lim & K.H. Lau and G. erectifrons
K.H. Lau, C.K. Lim & K. Mat-Salleh), bringing the current total to 15 taxa,
all highland species, found in hill, sub-montane and upper montane forests
ranging from 600 m to 2000 m a.s.l. Thirteen out of 15 of the known species
are believed to be hyper-endemic, found so far only in their respective type
localities.

Introduction

Geostachys (Baker) Ridl.is a relatively small genus within the Zingiberaceae
family, with only 21 species previously recorded. Its distribution ranges from
Vietnam, Thailand, Sumatera, Peninsular Malaysia and Borneo. Peninsular
Malaysia is the home for most of the species, with 15 taxa scattered in the
rain forest of this country (Holttum, 1950; Stone, 1980; Lau et al., 2005).
The name Geostachys was introduced by Baker (1892) as a subgenus
of Alpinia when he first described two species, A/pinia decurvata Baker and
A. secunda Baker, both from Perak. In his pioneering work, The Scitamineae
of The Malay Peninsula, Ridley (1899) elevated Geostachys as a genus, with
five species, adding three new ones: G. elegans Ridl., G. rupestris Ridl. and G.
penangensis Ridl. In 1920, Ridley described two other new species, namely,
G. primulina Ridl. and G. densiflora Ridl., bringing the total to seven. He had

130 Gard. Bull. Singapore 59 (1&2) 2007

earlier also described two taxa under separate genera: Carenophila montana
Ridl. in 1909 and Conamomum sericeum Ridl. in 1915, both of which were
subsequently transferred to Geostachys by Holttum (1950) in his important
monograph, The Zingiberaceae of The Malay Peninsula. Holttum further
added three new species: G. megaphylla Holttum, G. taipingensis Holttum
and G. tahanensis Holttum, all relatively unknown or not recollected until
recently. Stone (1980) discovered another taxon, G. /eucantha B.C. Stone,
making a total of 13, prior to our studies.

As a consequence of fieldwork to study the genus in their type
localities, all but G. montana (Ridl.) Holttum have been recollected, and
two new species found and published by the authors (Lau et al., 2005):
Geostachys belumensis C.K. Lim & K.H. Lau and G. erectifrons K.H. Lau,
C.K. Lim & K. Mat-Salleh.

Holttum’s account of Geostachys within the Zingiberaceae of
Peninsular Malaysia was made more than 50 years ago, and he intimated
that there were still several taxa based on incomplete data, also mentioning
that several species seemed rather closely allied, while other new species
may yet be discovered. We were encouraged to work on the revision of this
genus, to provide fresh data and updates on conservation status in the wild.
Further extension studies on the Bornean records of the genus may follow,
currently outside the scope of our study.

Studies on the Geostachys in other parts of the region have been
carried out by Gagnepain (1906), Valeton (1921) and Larsen (1962), in
which they had done various studies on this genus in Indo-China, Sumatera
and Thailand respectively.

Materials and Method

Field study and living plant collections

Observations were carried out during the recorded flowering and fruiting
season of the Geostachys species. Fieldwork was conducted at the type or
other known localities (Fig. 1) of each species to study and collect living
specimens and herbarium topotypes, and the in situ information proved
valuable in ascertaining characters and other attributes, such as coloration
and morphological variations, with reference to particular populations and/
or those in different localities. Field observations were also important to
analyze the habitat of each species.

Comparative morphology based on herbarium collections

Herbarium specimens or images of collections from the year 1884 to 2001 (in
addition, the authors’ recent collections) preserved at five major herbaria:
K, KEP, KLU, SING and UKMB, were scrutinized. A total of 98 specimens,

Taxonomic Revision of Geostachys in Peninsular Malaysia ia

including types were examined. Selected main morphology characters of
each species were investigated and compared. The characters include habit
of the plant, root and rhizome, leafy shoot, leaves, peduncle, stalk of cincinni,
bracts, pedicel, calyx, corolla tube, labellum, staminode, anthers, stamen, fruit
and seed where available.

200000 300000 500000 600000 700000

SCALE 1:3 000
+ +

800000

700000

600000

500000

South China
Sea

400000

200000

100000

200 Kilometer

200000 300000 400000 500000 600000 700000

Figure 1. Study areas at type localities of species of Geostachys.

132 Gard. Bull. Singapore 59 (1&2) 2007

Results and Discussion

Typification of the generic name Geostachys

Studies made on literature (Baker, 1892; Ridley 1899, 1920, 1924; Holttum,
1950) and as reported by Turner (2000) and Newman et al. (2004), have
shown that no type species has been designated for the genus to date. To
remedy this, and after due consultations, one of the two early taxa recognised
by Baker, Geostachys decurvata (Baker) Ridl., is proposed as the type
species. It displays all the essential characters of the genus, and can still be
conveniently referred to in its original type location at Bukit Larut, Perak.

GEOSTACHYS (Baker) Ridl., J. Straits Branch Roy. Asiat. Soc. 32 (1899)
158; Alpinia subg. Geostachys Baker, in Hook. f. Fl. Brit. India 6 (1892) 257.
TYPE: Geostachys decurvata (Baker) Ridl. (selected here).

A checklist and distribution of the Geostachys

Fourteen of the 15 species were found and studied by us in the field.
Geostachys montana could not be found in the type area, and remains
in doubt, as to whether it is truly distinct. A checklist of all the species in
Peninsular Malaysia is provided in Appendix 1.

All Geostachys species are highland species found in hill, sub-
montane and upper montane forests ranging from 600-2000 m a.s.l. in the
forest of Gunung Jerai (Kedah), Gunung Korbu (Perak), Gunung Ledang
(Johore), Gunung Mering (Malacca), Gunung Tapis (Pahang), Gunung
Benom (Pahang), Gunung Tahan (Pahang), Gunung Brinchang (Pahang),
Gunung Berembun (Pahang), Bukit Bendera (Penang), Bukit Larut (Perak),
Bukit Kedondong (Malacca), Bukit Fraser (Pahang), Genting Highlands
(Pahang), Cameron Highlands (Pahang), and on the hills of Semangkok
Forest Reserve (Selangor) and Belum Forest Reserve (Perak).

There have been records of G. penangensis from Borneo (Sarawak)
and some other Geostachys spp. in Sabah (C.K. Lim and K. Mat-Salleh, pers.
comm.). Initially, it was believed that G. penangensis was endemic to Penang
(Lau, 2004). However, the current revision only targetted on species from
Peninsular Malaysia. Nonetheless, the authors feel that further investigation
on the genus should also include other species from Borneo, as well as in
Thailand and Sumatera.

Morphological observations

Generally, all the species have stilt roots, or at least stilt roots-like coming
out from the rhizomes. Some species have true stilt roots, whereas some are
just having long and reticulated roots.

Taxonomic Revision of Geostachys in Peninsular Malaysia 133

As for the leaves, there are few characters that are quite useful
especially for field identification. The colours of the upper and lower surfaces
of the leaves are important as there are few species with maroon colour
underneath. The lamina comprises of four different types; widely elliptic,
narrowly elliptic, lanceolate and oblong. The other less prominent but useful
character is the presence of hairs at the ligule.

The structure of the inflorescence consists of two very different
types: decurved and erect. As for the decurved inflorescence, the curving of
the inflorescence starts at the peduncle and run through the whole rachis.
Each of the flowers curved at a very peculiar upwards manner, as if all of the
flowers are growing at one side only. However, for the erect type, the flowers
grow closely and in whorled-like manner. The flowers can either be single,
1-2 per cincinnus, 1-3 per cincinnus, or 1-5 per cincinnus.

The flowers of Geostachys can be either yellow or white. However,
majority of the species have yellow labellum with only 3 species having
white. Some of the species have flowers with crumpled margin whereby
some have smooth margin. Few species have staminodes on their flowers
and can be seen with red markings. Another distinct attribute that is quite
useful in recognizing between species is the presence of the anther crest.
The anther crest can be very prominent and 1s trilobed-like.

The shape of the fruits of this genus is either ovoid or ellipsoid.
The shape can be sometimes not so obvious, but in a particular species, the
shape is more or less constant. Table 1 summarizes the characteristic of each
species.

Endemism of Geostachys

Among the 15 recognised species, 13 are hyper-endemic to their respective
type localities. Some are observed as rare, and several species were found
only in few and small clumps at their habitats, e.g., G. primulina, G. tahanensis,
G. secunda, G. taipingensis, G. megaphylla, G. sericea, G. leucantha, G.
decurvata and G. belumensis. Attempts have been made to search for more
populations of these endemic species, but as yet to no avail. Adding to this
critical situation is the threat to their habitat. Except for G. tahanensis, G.
sericea and G. erectifrons whose habitats are within a National Park, those
of other species are rather exposed to hazards that entire populations could
be wiped out in a short period of time. As an example, the population of G.
taipingensis was no longer to be found on the second visit to a Known locality
near the type area. Conversely, however, other successful rediscoveries of the
Geostachys have been made, such as that of G. primulina after 80 years since
it was first collected (Lau, 2006 and C.K. Lim, pers. com.). A particular study
of the hyper-endemics, G. rupestris and G. penangensis, at their type areas
have shown that they are well able to survive under relatively protected

Gard. Bull. Singapore 59 (1&2) 2007

134

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situations. The two widespread species, G. elegans and G. densiflora, are
relatively common and not threatened.

Conservation status of Geostachys

A threat assessment on the genus is being carried out based on the IUCN
Red List Categories and Criteria Version 3.1 (IUCN, 2001) and Malaysia
Plant Red List (Chua and Saw, 2006). The final result of the assessment is
expected to be out together with the full revision of the Geostachys which is
currently in preparation by Lau ef al.

List of species of Geostachys in Peninsular Malaysia.

G. belumensis C.K. Lim & K.H. Lau
G. decurvata (Baker) Ridl.

G. densiflora Ridl.

G. elegans Rid.

G. erectifrons K.H. Lau, C.K. Lim & K. Mat-Salleh
G. leucantha B.C. Stone

G. megaphylla Holttum

G. montana (Ridl.) Holttum

G. penangensis Ridl.

G. primulina Rid.

G. rupestris Ridl.

G. secunda (Baker) Ridl.

G. sericea (Ridl.) Holttum

G. tahanensis Holttum

G. taipingensis Holttum

Acknowledgements

Thanks are due to Dato’ Musa Nordin, the Director-General, Department of
Wildlife and National Parks for assistance and encouragement for the trips
to Gunung Tahan, and to his field guides, Baharim Selat, Tayuddin Mohd.
Amin, and Juhari Basri; also to Imin Kamin of FRIM for field assistance.
Part of the research program was conducted at Tropical Forest Biodiversity
Centre (TFBC) in FRIM; the first author expresses his thanks to TFBC
Director, Dr. Saw Leng Guan, and to his staff for their support. Further
acknowledgements go to Dr. Lillian Chua and Hamidah Mamat, also from
FRIM for their assistance in the Red List Threat Assessment of the genus.
The second author acknowledges the contribution by his field team: Azizan
Busu, Alus Sarip and Zaidee Hitam. The first author’s research and fieldwork

Taxonomic Revision of Geostachys in Peninsular Malaysia i oe

were funded in part by IRPA Research Grant 09-02-02-EA0035. Further
thanks are due to the herbarium curators at K, KEP, KLU,SING and UKMB
for access to specimens, and also to the Penang Botanic Gardens.

References

Baker, J.G. 1892. Scitamineae. In J.D. Hooker, Flora of British India, vol. 6.
Reeve, London. 257pp.

Chua, L.S.L. and L.G. Saw. 2006. Malaysia Plant Red List. Guide for
Contributors. Forest Research Institute Malaysia, Malaysia.

Gagnepain, M.F. 1906. Zingibéracées nouvelles de l’herbier du Muséum.
Bulletin de la Société Botanique de France 53: 132-147.

Holttum, R.E. 1950. The Zingiberaceae of the Malay Peninsula. Gardens’
Bulletin, Singapore 13 (1): 224-236.

IUCN. 2001. Red List Categories and Criteria, Version 3.1. Gland,
Switzerland.

Larsen, K. 1962. Studies in Zingiberaceae 1. The genus Geostachys in
Thailand. Botanisk Tidsskrift 58: 43-49.

Lau, K. H. 2004. Observations on the endemic Geostachys (Baker) Ridl. of
Penang Hill and its environment. Folia malaysiana. 5: 109-114.

Lau, K.H., Lim, C.K. and K. Mat-Salleh. 2005. Two new species of Geostachys
(Zingiberaceae) from Peninsular Malaysia. Folia malaysiana 6: 83-94.

Lau, K.H. 2006. A ginger lost and found. Conservation Malaysia, FRIM
jo

Newman, M., Lhuillier, A & Poulsen, A, D, 2004. Checklist of the
Zingiberaceae of Malesia. Blumea Supplement 16. Nationaal Herbarium
Nederland.

Ridley, H.N. 1899. The Scitamineae of the Malay Peninsula. Journal of the
Straits Branch Royal Asiatic Society 32: 157-160.

138 Gard. Bull. Singapore 59 (1&2) 2007

Ridley, H.N. 1909. The Flora of the Telom and Batang Padang Valleys.
Journal of the Federated Malay States Museums 4(1): 1-78.

Ridley, H.N. 1915. Botany of Gunong Tahan. Journal of the Federated Malay
States Museums 6: 1-185.

Ridley, H.N. 1920. New and Rare Species of the Malayan Plants. Journal of
the Straits Branch Royal Asiatic Society 82: 1-201.

Ridley, H.N. 1924. The Flora of the Malay Peninsula. Vol. 4. London: Reeve
& Co, Lid,

Stone, B.C. 1980. A new Geostachys (Zingiberaceae) from Gunung Ulu Kali,
Pahang, Malaysia. Malaysian Journal of Science 6(A): 75-81.

Turner, I.M. 2000. The Plant Taxa of H.N. Ridley, 3. The Zingiberales. Asian
Journal of Tropical Biology 4(1), 1-47.

Valeton, T. 1921. Geostachys sumatrana. Bulletin du Jardin Botanique de
Buitenzorg Ser. IIT, 3: 146-147.

Gardens’ Bulletin Singapore 59 (1&2): 139-144. 2007 139

Materials Towards a Revision of Aulotandra Gagnep.
(Zingiberaceae)

M.F. NEWMAN

Royal Botanic Garden, 20A Inverleith Row
Edinburgh EH3 5LR, Scotland, UK

Abstract

Aulotandra Gagnep. has recently been transferred from the subfamily
Alpinioideae, tribe Alpinieae, to the subfamily Siphonochiloideae. Materials
towards a revision of Aulotandra and Siphonochilus J.M.Wood & Franks
are presented.

Introduction

Harris et al. (2006) have included Aulotandra Gagnep. in a phylogenetic
analysis in order to determine its correct place in the new classification of
Zingiberaceae by Kress et al. (2002).

Aulotandra was, until recently, the only African genus of
Zingiberaceae which had not been included in a molecular systematic study.
Two molecular datasets, chloroplast and nuclear, placed Aulotandra closest
to Siphonochilus JM. Wood & Franks, showing that genetic divergence
levels were smaller between accessions of Au/otandra and Siphonochilus
than between Aulotandra and any other taxon included in the analysis.

In addition, phylogenetic analyses of the two data matrices showed
that the species of Aulotandra and Siphonochilus sampled in that study
formed a monophyletic group. It was clear from the shared synapomorphies
and the high branch support for the clade containing these genera that this
relationship was very close. The two data sets showed some discrepancy as
to the relationships between these two genera - the ITS analysis indicating
that Aulotandra was monophyletic, but the rnL-F results suggesting that
the two genera were paraphyletic.

Accepting that Aulotandra and Siphonochilus form a monophyletic
group led to the transfer of Aulotandra from subfamily Alpinioideae, tribe
Alpinieae, to subfamily Siphonochiloideae. Taking this study further, it
is clear that the species in subfamily Siphonochiloideae must be revised
together.

140 Gard. Bull. Singapore 59 (1&2) 2007

Materials and Methods

A list of the names in Aulotandra, Siphonochilus and Kaempferia L. in
Africa was compiled using the International Plant Names Index (www.ipni.
org) and the World Checklist of Monocotyledons (http://www.kew.org/wcesp/
home.do). Protologues were consulted and details of the type of each name
were added. Where possible, the collector, collection number and herbarium
location are given but, in some cases, it is not clear from the literature where
types are to be found.

Results

In total, there are 30 names to be revised in the Siphonochiloideae, eight
in Aulotandra, 12 in Siphonochilus, and 10 in Kaempferia. The majority of
names are based on type specimens held at the Muséum national d’Histoire
naturelle, Paris. A few are yet to be located; those collected by German
botanists may have been lost.

Names in Aulotandra Gagnep.

1. Aulotandra angustifolia H. Perrier, Bull. Soc. Bot. France 86: 178. 1939.
Type: Perrier de la Bathie 7264 (P).

2. Aulotandra humbertii H. Perrier, Bull. Soc. Bot. France 86: 180. 1939.
Type: Humbert s.n. (P).

3. Aulotandra kamerunensis Loes., Bot. Jahrb. Syst. 43: 389. 1909.
Type: Zenker 3696 (US, WU, WRSL).

4. Aulotandra madagascariensis Gagnep., Bull. Soc. Bot. France 48: LX XIX.
1901.
Type: Humblot 448 (P).

5. Aulotandra trialata H. Perrier, Bull. Soc. Bot. France 86: 181. 1939.
Type: Perrier de la Bathie 1021 (P).

6a. Aulotandra trigonocarpa H. Perrier var. trigonocarpa. Bull., Soc. Bot.
France 86: 179. 1939.
Type: Perrier de la Bathie 19014 (P).

6b. Aulotandra trigonocarpa H. Perrier var. calcicola H. Perrier, Bull. Soc.
Bot. France 86: 180. 1939.
Type: Perrier de la Bathie 1672, 1687 (P).

6c. Aulotandra trigonocarpa H. Petrier var. gypsicola H. Perrier, Bull. Soc.
Bot. France 86: 180. 1939.
Type: Perrier de la Bathie 15943 (P).

Materials Towards a Revision of Aulotandra 141

Names in Siphonochilus JM. Wood & Franks

1. Siphonochilus aethiopicus (Schweinf.) B.L.Burtt, Notes Roy. Bot. Gard.
Edinburgh 40(2): 372. 1982.
Type: Cienkowski s.n., Steudner s.n.

2. Siphonochilus bambutiorum A.D.Poulsen & Lock, Kew Bull. 54: 203.
eles)
Type: Poulsen & Liengola 1146 (holo, C; iso BR, E, K, MO).

3. Siphonochilus brachystemon (K.Schum.) B.L.Burtt, Notes Roy. Bot. Gard.
Edinburgh 40(2): 372. 1982.
Type: Volkens 201 (B), Holst 3100 (B).

4. Siphonochilus carsonii (Baker) Lock, Kew Bull. 39: 841. 1984.
Type: Carson s.n. (K).

5. Siphonochilus decorus (Druten) Lock, Kew Bull. 54: 346. 1999.
Type: Schweickert s.n. (PRE).

6. Siphonochilus evae (Briq.) B.L.Burtt, Notes Roy. Bot. Gard. Edinburgh
40(2): 372. 1982.
lype: Prosch.J2 (G).

7. Siphonochilus kilimanensis (Gagnep.) B.L.Burtt, Notes Roy. Bot. Gard.
Edinburgh 40(2): 372. 1982.
Type: Le Testu s.n. (P).

8. Siphonochilus kirkii (Hook.f.) B.L.Burtt, Notes Roy. Bot. Gard. Edinburgh
40(2): 372. 1982.
Type: Kirk s.n. (K).

9. Siphonochilus natalensis (K.Schum.) J.M.Wood & Franks, Natal pl. 6(3):
plates 560-561. 1911.
Type: Wood 544 (K).

10. Siphonochilus nigericus (Hepper) B.L.Burtt, Notes Roy. Bot. Gard.
Edinburgh 40(2): 372. 1982.
Type: Dalziel 276 (K).

11. Siphonochilus parvus Lock, Kew Bull. 46: 269. 1991.
Type: Congdon 46 (K).

12. Siphonochilus rhodesicus (T.C.E.Fr.) Lock, Kew Bull. 39: 841. 1984.
Type: Fries 1146 (UPS).

Names in Kaempferia L. in Africa

1. Kaempferia aethiopica (Schweinf.) Benth. var. angustifolia Ridl., J. Bot.
ZOASMe 18ST.
Type: Welwitsch 683 (K).

2. Kaempferia ceciliae N.E.Br. Bull, Misc. Inform. 169. 1906.
Type: Cecil 248 (K).

3. Kaempferia dewevrei De Wild. & T. Durand, Bull. Soc. Roy. Bot. Belgique

142 Gard. Bull. Singapore 59 (1&2) 2007

38: 142. 1899.
Type: Dewevre 1021.

4. Kaempferia ethelae JM. Wood, Gard. Chron. 23: 94. 1898.
Type: not known.

5. Kaempferia homblei De Wild., Repert. Spec. Nov. Regni Veg. 13: 195.
1914
Type: Homblé 851, 907.

6. Kaempferia montagui F.M. Leight., S. African Gard. 22:57, 59. 1932.
Type: Montagu 888/21 (K).

7. Kaempferia pleiantha K. Schum., Bot. Jahrb. Syst. 15(4): 425. 1892.
Type: Buchner 694, Mechow 559b.

8. Kaempferia puncticulata Gagnep., Bull. Soc. Bot. France 53: 353. 1906.
Type: Kiener s.n. (P).

9. Kaempferia stenopetala K. Schum., Pflanzenr. IV 46 (Heft 20): 69. 1904.
Type: Wood 1942.

10. Kaempferia zambesiaca Gagnep., Bull. Soc. Bot. France 53: 355. 1906.
Type: Le Tesm 363 (8).

Recommendations

Preliminary morphological observations and the sequence results presented
by Harris et al. (2006) suggest that there may be only one genus in subfamily
Siphonochiloideae. In order to test this hypothesis, a molecular and
morphological study with wider sampling should be carried out to determine
the relationships between the species and assess the limits of these two
genera. All names listed above should be revised so that the number of
accepted species, their distributions and their conservation status may be
confidently known.

Acknowledgements

I am grateful to my colleagues, David Harris, Michelle Hollingsworth,
Michael Moller and Alexandra Clark, for allowing me to present their
results and to build upon them.

References

Harris, D.J., M.F. Newman, M.L. Hollingsworth, M. Moller and A. Clark,
2006. The phylogenetic position of Aulotandra Gagnep. (Zingiberaceae).
Nordic Journal of Botany 23(6): 725-734.

Materials Towards a Revision of Aulotandra 143

Kress, W.J., L.M. Prince and K.J. Williams, 2002. The phylogeny and a new
classification of the gingers (Zingiberaceae): evidence from molecular
data. American Journal of Botany 89: 1682-1696.

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Gardens’ Bulletin Singapore 59 (1&2): 145-172. 2007 tAS

Etlingera Giseke of Java
A.D. POULSEN

Royal Botanic Garden Edinburgh, 20A Inverleith Row
Edinburgh EH35LR, Scotland

Abstract

Nine species of Etlingera Giseke are known in Java, though two of them
have not been collected recently. An identification key is given, along with
descriptions, illustrations, and notes on local names, uses, and ecology. The
conservation status of each species is assessed. Two species remain enigmatic
and the remaining seven, including FE. parva, which is synonomized with E.
brachychila, are all found in Borneo, Sumatra and/or the Malay Peninsula.

Introduction

The genus Etlingera Giseke (Zingiberaceae) is an Indo-Pacific genus
especially rich in species in the perhumid forests of Thailand, Malaysia,
Indonesia and New Guinea. Many species are useful to man as food,
condiment, medicine or as ornamentals. They also play an important role in
the understorey as a food source for animals. The leafy shoots can be up to
8 m tall and often dominate gaps in disturbed forests.

In Java, Etlingera may be confused with other genera that have
radical inflorescences: Amomum Roxb., Hornstedtia Retz. and Zingiber
Mill. Etlingera is, however, distinguished from all these by having a staminal
tube.

Blume (1827), described five ginger species from Java which are
included in the present paper as Etlingera coccinea (Blume) S. Sakai &
Nagam., E. foetens (Blume) R.M.Sm., E. hemisphaerica (Blume) R.M.Sm.,
E. solaris (Blume) R.M.Sm.,an E. walang (Blume) R.M.Sm. At that time, E.
elatior (Jack) R.M.Sm. had already been collected in Sumatra and described.
Subsequently, E. megalocheilos Griff. was described from Peninsular
Malaysia (Griffith, 1851) and Valeton (1904) documented its presence in
Java with useful information also on other species of Etlingera there. Valeton
published further observations and clear and informative illustrations in
1906. In 1921, he described what is here included as E. heyniana (Valeton)
R.M.Sm. and E. parva (Valeton) R.M.Sm. Since then no further species of

146 Gard. Bull. Singapore 59 (1&2) 2007

Etlingera has been described from Java and thus the revision by Bakhuizen
f. (1958; 1968) includes a total of nine species. In the present treatment, seven
of these at least are good species but two remain somewhat ”mysterious”.

Material and Methods

Measurements of plants follow Poulsen (2006). Only recent collections
are cited in the present paper. As new flowering material will be collected
allowing detailed measurements, a wider range can be expected for some
of the character measurements included below. This is demonstrated by the
variation exhibited by the most commonly collected E. coccinea, the species
with most collections assessed in the present paper.

Assessments of conservation status were carried out following
IUCN (2000), based on current knowledge and using their terminology on
categories, criteria and subcriteria.

Key to species of Etlingera in Java

1. Ligule 4-8 cm long, deeply bilobed; fruit beaked ....... ee. 7. E. solaris
1. Ligule <3.5 cm, + entire; fruit rounded, flat-topped or with depressed
APIOK nsisse ccesnliadal aoa dro teawan on cnmabeltleaa daha nee ne te eee teeta ss ee ge ae 2
2. Inflorescence spike raised more than 10 cm above ground; peduncle
extended avove Sround, F CLECE ca Nesccscsdtendeee ehereeoate eee mena ee 3
2. Inflorescence spike at ground level or partly embedded in soil; peduncle
SUDTSTHAMCAN Aare kee icen cen neh see ce oe eecee nett ere -

3. Leaves green beneath; inflorescence erect, 60-200 cm; bracts to 13 cm
long, outer ones reflexed when flowering; receptacle extended to 10 cm
eee ties bei da in 8 avian trnedends Semi teed «ek geet nah ptt atta! 3. E. elatior

. Leaves reddish beneath (especially when young); inflorescence erect,
15-100 cm; outer bracts forming a cup-shaped spike not recurved when
flowering) receptacle = Cnn nr. cee teeter eee ee 5. E. hemisphaerica

ass

. Labellum short (< 15 mm), extended <5 mm beyond stigma; fruit with soft
Fond Wi pee trebe dental ebb heir hr stn tts ire, dined hon Sahar Hoes cae any sac 1. E. brachychila
Labellum long (> 35 mm), extended >7 mm beyond stigma; fruit smooth,
ridged or roughly papmlose - 3.2 l A edereccsccscabee eects teaen hee eee 5

. Labellum extended <32 mm beyond anther; filament <1 mm long...............
PPLE ESR EA DR Mite Mot atch ri Paden materia Ha ode nse sa9- 4. E. foetens
. Labellum extended >34 mm beyond anther; filament >3 mm long. ......... 6

Etlingera Giseke of Java 147

6. Petiole usually absent; dorsal corolla lobe hooded over the anther; labellum
yellow with red margin; anther dehiscing from 1.5 mm above base to
apex; fruit flat-topped with roughly papillose ridges ....... 2. E. coccinea

6. Petiole 1-4 cm; dorsal corolla lobe not covering the anther; labellum red
with yellow margin; anther dehiscing in upper half only; fruit top rounded,
smooth or with’ ate w Wartsa nasa Wisse 6. E. megalocheilos

1. Etlingera brachychila (Ridl.) R.M. Sm.

Notes: Roy. Bot. Gard. Edinburgh 43 (1986) 243; Smith, Notes Roy. Bot.
Gard. Edinburgh 43 (1986) 452; Poulsen, Etlingera of Borneo (2006) 66.
Basionym: Hornstedtia brachychila Ridl., J. Straits Branch Roy. Asiat. Soc.
46 (1906) 239. Type: Malaysia, Sarawak, Bau, July 1903, flowering, H.N.
Ridley s.n. [lecto, K (designated by Turner (2000): 34)].

- Etlingera parva (Valeton) R.M. Sm., Notes Roy. Bot. Gard. Edinburgh 43
(1986) 248, syn. nov. Basionym: Geanthus parvus Valeton, Bull. Jard. Bot.
Buitenzorg ser. 3,3 (1921) 145. - Amomum parvum (Valeton) Bakhuizen f.,
Bekn. FI. Java 18 (1958) 30; Bakhuizen f.in Backer, Fl. Java 3 (1968) 56. Type:
Indonesia, Java, Cipatuja, Bivak Denu, 450 m, 20 Aug 1913, C.A. Backer
8887 (holo, BO).

Rhizome long-creeping (80 cm between neighbouring leafy shoots); stilt
roots absent. Leafy shoot to 3 m, with up to c. 30 leaves; base to 3 cm in
diameter, bright red. Sheath striate, margin membranous and glabrous.
Ligule to 9.5 mm, entire, truncate to slightly emarginate, greenish, glabrous,
margin membranous. Petiole 10-20 mm, hirsute especially adaxially and at
base. Lamina oblong to narrowly obovate, to 55 x 9 cm, smooth, green, pale
beneath, with 0.5 mm long white hairs with swollen base along midrib and
near base above; glabrous beneath; average length to width ratio (4—)6(-8);
base rounded to cuneate, + unequal; apex acuminate 1.5 cm. Inflorescence
(including peduncle) to 17 cm, embedded in the soil, arising from base or
along rhizome, with up to 29 flowers, 5 open at a time. Peduncle to 10 cm,
subterranean, ascending, peduncular bracts to 2.5 x 1.8 cm, base with pale
hairs, upper just overlapping the base of the spike. Spike 7 x 3 cm, cylindrical,
flowers extended 5 cm above the bracts, length only including bracts: 2.5 cm.
Sterile bracts 2, distichous, 2.5 x 1.2 cm, elliptic, + acute, brownish, pubescent
at base. Fertile bracts 2.2—2.5 x 0.3-0.7 cm, narrowly spathulate, boat-shaped,
apex rounded, pale brown, pubescent at very base only. Bracteole 2.1 cm, pale
reddish, with two fissures of 0.5—1.5 cm, pubescent near base, apex bilobed
with a few hairs. Flower: Calyx 4.5—6.1 cm, reaching base of filament shorter
than corolla lobes, red, with one fissure of ca 3 cm, glabrous, apex + tridentate
with 3 mucro <1 mm. Corolla tube 5.7 cm, red, glabrous outside and inside.

148 Gard. Bull. Singapore 59 (1&2) 2007

Lobes red, with a few hairs near apex; dorsal lobe 19-20 x 6-7 mm, almost
reaching middle or apex of anther, elliptic, cucullate, margin inrolled, apex
rounded; lateral lobes 20-21 x 5 mm, narrowly elliptic, cucullate, margin
inrolled, apex rounded; insertion slightly oblique, diverging, 3 mm below
dorsal lobe. Staminal tube 10 mm. Labellum broadly ovate, 3-lobed, 14 x 16
mm, red to dark orange, lateral lobes rigid, erect on either side of the stamen
(pushing the dorsal lobe apart and exposing the stamen), margin recurved,
especially in the distal part, yellow, central lobe rigid, entire, rounded, red
or orange, extended 2.5 mm beyond anther. Stamen 11 mm; filament 4-4.5
mm, with a few hairs on the margin especially near base; anther 7.5 x 3.5-5.5
mm, erect, rectangular, red, slightly wider at apex, apex hairy, anther crest
truncate; thecae dehiscing ca 3.5 mm in the middle (from 2.5 mm above
base to ca 1.5 mm below apex), pubescent, especially below the slits. Style ca
6.5 cm, hairy dorsally near apex. Stigma 3.5-4 mm wide, transverse narrow-
elliptic with scattered hairs, red; ostiole transverse 2 mm, facing downwards.
Ovary 4 x 3.5 mm, densely sericeous; epigynous glands 4 mm, deeply lobed,
each half irregularly lobed. Infructescence: head 6 cm, subglobular, ca 16
fruits per head; fruit 2.5 x 2.5 cm, pyriform with soft, spiny teeth up to 7 mm
long, especially developed on the top, dark purple-brown, pubescent. Seeds
rounded-angular up to 4mm across. Plate 1A.

Local names and uses: None documented.
Etymology: The epithet refers to the short labellum.
Ecology and habitat: Primary lowland (250-450 m) forest.

Distribution: W Java and Borneo.

Conservation status: EN Blab (111). Deforestation seriously threatens the
forest in the southern part of Java, including its type locality at Cipatuja. With
only one recent collection from one location, despite intensive searching, it
is potentially endangered in Java. Valeton noted as early as 1921(b), that this
species is "apparently very rare as are the primaeval forests, its habitat.’

Additional material examined: Banten Province, buffer zone of Ujung Kulon
NP, Gunung Honje, 250 m, 17 Nov 1996, flowering and fruiting, Funakoshi
IU 19 (E, BO n.v.).

Notes: The collection by Funakoshi is to my knowledge the only one from
Java since the type of E. parva was collected in 1913, and the first including
the infructescence. The material matches the type of EF. brachychila from

Etlingera Giseke of Java 149

Sarawak very well, except the calyx and corolla tube being slightly longer
and the lateral lobe of the labellum being yellow but these characters are
still within the range of FE. brachychila var. vinosa A.D. Poulsen — also from
Borneo.

Etlingera brachychila is easily distinguished from other Javanese
species with their inflorescence at ground level by its poorly developed
involucral bracts, short labellum and its conspicuous soft-toothed fruit
somewhat resembling an Amomum. It resembles two species so far only
known from Borneo: FE. aurantia A.D. Poulsen and E. kenyalang A.D.
Poulsen & H. Chr. Etlingera brachychila is distinguished from the other two
by the smaller and fewer sterile bracts which give less support to the spike.
The apex of the calyx is less mucronate. Finally, E. brachychila differs in
its flower colour and the orientation of the lateral lobes of the labellum
(reflexed vs. erect or involute).

In Borneo a variety FE. brachychila var. vinosa has a white-waxy
sheath and often more or less bullate leaves which are burgundy beneath.
In Java, this variation in leaf colour has not been documented and the leaves
are only sparsely hairy. Future collections may demonstrate more pubescent
leaves like those E. brachychila var. brachychila in Borneo.

2. Etlingera coccinea (Blume) S. Sakai & Nagam.

Edinburgh J. Bot. 60 (2003) 190; Poulsen, Etlingera of Borneo (2006) 88.
Basionym: Elettaria coccinea Blume, Enum. Pl. Javae (1827) 53; Hasskarl,
Pl. Jav. Rar. (1848) 134; Burtt & Smith, Notes Roy. Bot. Gard. Edinburgh 31
(1972) 212. - Geanthus coccineus Reinw., nom. nud., Catalogus (1823) 29.

- Alpinia coccinea (Blume) D. Dietr., Syn. Pl. 1 (1839) 12. - Cardamomum
coccineum (Blume) Kuntze, Revis. Gen. Pl. 2 (1891) 686. - Amomum
coccineum Benth. [Gen. pl. 3 (1883) 644, nom. inval.| ex Schumann, Bot.
Jahrb. Syst. 27 (1899) 305; Valeton, Bull. Inst. Bot. Buitenzorg 20 (1904) 44;
Valeton, Icon. Bogor. 2 (1906) t. 156, t. 157 figs. 21-22; Bakhuizen f., Bekn. FI.
Java 18 (1958) 34; Bakhuizen f. in Backer, Fl. Java 3 (1968) 57; van Steenis,
Mt. Flora of Java, (1972) pl. 57: 2. - Achasma coccineum (Blume) Valeton,
Bull. Inst. Bot. Buitenzorg 20 (1904) 93; Heyne, Nuttige pl. Nederl. Indié 1
(1927) 489. Type: Probably Java, C.G.C. Reinwardt s.n., bar code L0193260,
accession number 905339126, including only fertile material to the right of
the leaf (lecto, L, designated by Sakai & Nagamasu, 2003).

- Achasma macrocheilos Griff., Not. Pl. Asiat. 3 (1851) 429, Ic. Pl. Asiat. 3
(1851) t.357.- Amomum macrocheilos (Griff.) Baker, Fl. Brit. India 6 (1892)
235. - Hornstedtia macrocheilos (Griff.) Ridl., J. Straits Branch Roy. Asiat.
Soc. 32 (1899) 147; Schumann, Pflanzenr. IV, 46 (1904) 198; Ridley, Matr. FI.

150 Gard. Bull. Singapore 59 (1 &2) 2007

Mal. Penin. 2 (1907) 39; Ridley, Fl. Mal. Penin. 4 (1924) 271. Type: Malaysia,
Malacca, Ayer Punnus, W. Griffith s.n. (not seen, specimen probably lost).

- Amomum gomphocheilos Baker, Baker, Fl. Brit. India 6 (1892) 236. Type:
Malaysia, Perak, H. H. Kunstler 1897 (holo, K).

- Hornstedtia punicea (Roxb.) K. Schum., Pflanzenr. IV, 46 (1904) 197, pro
parte, not including type; Etlingera punicea auct. non (Roxb.) R.M. Sm.:
Smith, Notes Roy. Bot. Gard Edinburgh 43 (1986) 443; Cowley in Coode
et al., Checklist Pl. Brunei (1996) 419; Keng et al., Concise Fl. Singapore 2
(1998) 57; Khaw, Gard. Bull. Singapore 53 (2001) 229.

- Hornstedtia winkleri Ridl., Bot. Jahrb. Syst. 44 (1910) 530. Type: Indonesian
Borneo, SE of Sungai Sibak in Gunung Benanpin, 12 Aug 1908, flowering,
Hubert J.P. Winkler 3175 (holo, WRSL).

Rhizome l\ong-creeping (70-100 cm between neighbouring leafy shoots),
2.5-4 cm diam., scales to 6 cm long, ensheathing. Leafy shoot 5-8 m, with
up to to 32 leaves; base to 8-12 cm diam., green or reddish brown. Sheath
green, yellowish green or brownish, striate, + reticulate (especially when
young), glabrous or slightly pubescent (especially on cross ribs), margin
ciliate. Ligule 10-15 mm, entire, green to purplish brown, + pubescent.
Lamina sessile (rarely with a 1-2 cm, petiole-like, winged-attenuate base),
oblong to narrowly obovate, to 130 x 23 cm, mid- or dark green, young
leaf reddish brown beneath, glabrous (rarely pubescent beneath); average
length to width ratio 4-6; base cuneate, sometimes irregularly winged; apex
acuminate 1.5 cm. Inflorescence (including peduncle) to 47 cm, arising
from the rhizome near base of leafy shoot, with 15-27 flowers, 4-14 open
at a time. Peduncle 2—33 cm, subterranean, peduncular bracts cream to pale
brown. Spike 8-10 x 3-5 cm, ovoid to cylindrical, flowers extending 24
cm above the bracts, length only including bracts: 6-8 cm. Sterile bracts:
4-5 forming a dense support, distichous (uppermost one sometimes in the
middle), 4-6 x 1.5—3 cm, + pale reddish brown or cream with + reddish apex
and pale brown margin, pubescent near base. Fertile bracts 4~7 x 0.7-1.5
cm, spathulate, membranous, cream, pale red or brown especially at apex,
pubescent especially near base and apex. Bracteole 3.8—5 cm, pale red at
least at apex, with two fissures of 6-15 mm, + pubescent, apex 2-toothed,
ciliate. Flower. calyx 7-8.3 cm, reaching base of anther and shorter than
corolla lobes, pale pink with darker apex, with one fissure of 2.5-4 cm,
glabrous, apex 3-toothed. Corolla tube 4.9-6.8 cm, cream, glabrous outside,
tube inside with an irregular band c. 1 cm 2 cm below labellum; lobes reddish
pink, glabrous, reaching beyond anther; dorsal lobe 21-31 x 9-11 mm, elliptic,

Etlingera Giseke of Java 151

apex rounded, hooded over the anther; lateral lobes 21—25 x 4-7 mm, elliptic,
apex rounded, insertion oblique and converging; staminal tube 4-12 mm.
Labellum 3-lobed, 50-65 x 21-25 mm, red with yellow in centre, glabrous,
lateral lobes folded over stamen, pale to dark red at margin, margin finely
plicate, central lobe c. 30 x 16 mm, spathulate, emarginate to 15 mm, rarely
entire, dark red extended 40-50 mm beyond anther. Stamen 12 mm long:
filament 3-7 x 3.5-5 mm (widest at base), white to pale red; anther 9-10 x
2.5-5.5 mm (widest at apex), emarginate to 2.5 mm, angled 120 , pink; thecae
dehiscing from 1.5 mm above base to apex, glabrous. Style 8—8.5 cm, sparsely
pubescent, flexistylous. Stigma 2.5 mm wide, white or pale pink, triangular to
heart-shaped, + hairy, ostiole apical, transverse. Ovary 5 mm long, densely
pubescent; epigynous glands 5-7 mm long, bipartite, linear. Infructescence:
head to 12 cm, globose, bracts persistent, 5—15 fruits per head, fruit 4.5 x 3.5
cm, pyriform, flat-topped with irregular radiating roughly papillose ridges
up to 6 mm high, brownish (red when young), pubescent. Seeds 2-3 mm
across. Plate 1B.

Local names and uses: Blume (1827) listed tepus, tepus gede, and mancirian
(Sundanese). Valeton (1904) mentioned that tepus bener (genuine) and
tepus leuaweung (forest or wood) as names for subspecies with an entire or
emarginate apex to the labellum, respectively. Heyne (1927) also mentioned
tepus bener but specified that mancirian is the name of the flower, and rongod
refers to the fruit.

The young leafy shoot and the fruits are edible (Poulsen et al. 2282).
Shoot tastes cabbage-like when young; bitter when old. Even though it is
often considered useful I never came across it being planted and cultivated
in Java.

Etymology: Coccinea means scarlet.

Ecology and habitat: Old field edges, in traditional home gardens (but not
planted), secondary forests, or in gaps or near streams of primary forests at
40-1650 m. Valeton (1904) noted that the seeds are frequently dispersed by
animals that leave behind a big hole in the top of the empty fruit.

In Sumatra and Borneo, EF. coccinea is pollinated by bees or
spiderhunters (Kato et al., 1993; Sakai et al., 1999). Fruit eaten by rodents.

Distribution: Thailand, Peninsular Malaysia, Singapore, Sumatra, Java and
Borneo.

Conservation status: LC (least concern), because of its wide distribution and
persistence in very disturbed habitats.

152 Gard. Bull. Singapore 59 (1&2) 2007

Additional material examined: Banten Province: bufferzone of Ujung Kulon
NP, Gunung Cibiug, 3 km E of Tamanraya village (6 47’S 105 32’E), 130
m, 27 Apr 2005, flowering and fruiting, Poulsen et al. 2343 (AAU, BO, E).
West Java Province: Gunung Salak (6 40’S 106 44’E), 950 m, 28 Sep 2003,
flowering, Poulsen et al. 2233 (BO);Halimun NP, Citalahab (6 44’S 106 32’E),
1100 m, 20 Mar 2004, flowering and fruiting, Poulsen et al. 2282 (AAU, BO,
E); Gunung Kancana, 2 km WSW of Parabuan village (6 55’S 107 03’E), 950
m,27 Mar 2004, flowering, Poulsen et al. 2294 (AAU, BO); Cisarakan (7'05°S
106 35’E), 500 m, 10 Aug 2006, flowering, Poulsen et al. 2457 (BO); Cikaso
(7 22’S 106 37°E), 40 m, 11 Aug 2006, flowering, Poulsen et al. 2458 (BO, E);
Gunung Cikuray (7 19’S 107'54’E), 1250 m, 13 Aug 2006, flowering, Poulsen
et al. 2463 (BO, E).

Notes: In Java, E. coccinea can most easily be confused with E. megalocheilos.
Etlingera coccinea differs in its (most often) sessile leaves, absence of tufts
on the apex of the calyx, the broad, pink dorsal corolla lobe hooded over
the stamen, the long-elongate labellum with a broad yellow centre and red
margin and thecae dehiscing almost to base. The margins are inrolled and
like a tube so that the red at first glance appears to be in the centre of the
labellum (like in E. megalocheilos).

Most often the leaf of FE. coccinea is glabrous but not always. During
my recent survey, one plant was collected in which the lamina was pubescent
beneath (Poulsen et al. 2457) and E. megalocheilos also varies regarding
the indumentum beneath the lamina. Thus as demonstrated in the Bornean
revision of Etlingera (Poulsen, 2006) indumentum is not a completely
reliable character.

Schumann (1904) regarded E. coccinea as a synonym of E. punicea,
and therefore the latter name was used by Smith (1986) and others. This
misconception was finally sorted out by Sakai & Nagamasu (2003).

Valeton (1904) described beautifully how the flowers in one
inflorescence opened, in up to three circles of up to 15 flowers, each lasting
only one day; he obviously took great care in making precise observations
of live plants of Etlingera in Java. Valeton (1904) further mentioned that
the ’subspecies’ (cf. etymology above) with an entire apex to the labellum is
much more common than the emarginate one, whereas I found the opposite
to be the case. In any case, I do not see any reason to formally recognize this
variation in two subspecies.

The local name in Java, tepus (Sundanese), is also used in Borneo by
many tribes (Poulsen, 2006). Often peoples in Borneo use a generic name
for all gingers that seems to be derived from that. The giant form that I
collected in several places in Borneo is most similar to wild plants in Java,

Etlingera Giseke of Java 153

such as those on Gunung Salak. All these lack a strong smell. Many local
names in Borneo include an epithet that refers to the distinct smell. Some
forms may have been selected over centuries and taken into cultivation.
These plants are often smaller, but I did not find any floral measurements
justifying the recognition of the smaller and smellier plants even at a lower
taxonomic rank.

In this context it is interesting that the enigmatic Etlingera walang
(Blume) R.M. Sm. is considered a very smelly plant. New collections in
Java of a smelly Etlingera coccinea may provide the necessary evidence for
establishing this synonymy.

3. Etlingera elatior (Jack) R.M. Sm.

Notes Roy. Bot. Gard. Edinburgh 43 (1986) 244; Smith, Notes Roy. Bot.
Gard. Edinburgh 43 (1986) 442; Lim, Folia Malaysiana 1 (2000) 4; Lim, Folia
Malaysiana 2 (2001) 168; Khaw, Gard. Bull. Singapore 53 (2001) 206; Sakai
& Nagamasu, Edinburgh J. Bot. 60 (2003) 192452; Poulsen, Etlingera of
Borneo (2006) 111. Basionym: A/pinia elatior Jack, Descriptions of Malayan
plants 2 (7) (1822) 2; Miquel, Fl. Ned. Ind., 1. Suppl. (1861) 273. - Nicolaia
elatior (Jack) Horan., Prodr. Monogr. Scitam. (1862) 32; Burtt & Smith,
Notes Roy. Bot. Gard. Edinburgh 31 (1972) 210, fig. 17B. Type: Nias Island
or Ayer Bangy, W coast of Sumatra, W. Jack s.n. 1818 (specimen lost). Bunga
kenchong (Malay).

- Geanthus speciosus Reinw., Catalogus (1823) 29, nom. nud.

- Diracodes javanica Blume, Enum. Pl. Javae (1827) 55. - Alpinia javanica
(Blume) D. Dietr., Syn. Pl. 1 (1839) 13. - Alpinia acrostachya Steud., Nomencl.
Bot. 2 (1840) 62. - Alpinia diracodes Loes., Nat. Pflanzenfam. ed.2,15,A (1930)
614. Type: Java, Hariang, C.G.C. Reinwardt s.n., bar code L0041105 (holo, L).

- Elettaria speciosa Blume, Enum. Pl. Javae (1827) 51; Hasskarl, Pl. Jav. Rar.
(1848) 133; Valeton, Bull. Inst. Bot. Buitenzorg 20 (1904) 29.

- Alpinia speciosa (Blume) D. Dietr., Syn. Pl. 1 (1839) 13. - Nicolaia speciosa
(Blume) Horan., Prodr. Monogr. Scitam. (1862) 32; Valeton, Bull. Jard. Bot.
Buitenzorg ser. 3,3 (1921) 138; Heyne, Nuttige pl. Nederl. Indié 1 (1927) 488;
Bakhuizen f., Bekn. Fl. Java 18 (1958) 47; Bakhuizen f. in Backer, Fl. Java 3
(1968) 64. - Phaeomeria speciosa (Blume) Koord., Exkurs.-Fl. Java 1 (1911)
332; Merrill, Enum. Phil. Pl. 1 (1925) 241; Holttum, Gard. Bull. Singapore 13
(1950) 181. Type: Java, in forest near Kapang Dungan, H. Kuhl & J.C. van
Hasselt s.n., bar code LO193778 (lecto, L, designated by Sakai & Nagamasu,
2003).

154 Gard. Bull. Singapore 59 (1&2) 2007

- Alpinia magnifica Roscoe, Monandr. PI. Scitam. (1828) t. 75; Hooker, Bot.
Mag. 59 (1832) t. 3192. - Bojeria magnifica Raf., Fl. Tellur. (1836) 50, nom.
inval. - Phaeomeria magnifica Lindl., Intr. Nat. Syst. Bot., ed. 2 (1836) 446,
nom. inval. - Nicolaia imperialis Horan., Prodr. Monogr. Scitam. (1862) 32, t.
1,nom. inval.- Amomum magnificum (Roscoe) Benth. [Gen. P1.3 (1883) 644,
nom. inval.] ex Schumann, Bot. Jahrb. Syst. 27(1899) 307; Valeton in Merrill,
Interp. Herb. Amboin. (1917) 159. - Cardamomum magnificum (Roscoe)
Kuntze, Revis. Gen. Pl. 2 (1891) 687. - Hornstedtia imperialis (Horan.) Rid1.,
J. Straits Branch Roy. Asiat. Soc. 32 (1899) 148. - Nicolaia magnifica (Roscoe)
K. Schum. ex Valeton, Bull. Inst. Bot. Buitenzorg 20 (1904) 35. - Phaeomeria
magnifica (Roscoe) K. Schum., Pflanzenr. IV, 46 (1904) 262; Loesener, Nat.
Pflanzenfam. ed. 2, ISA (1930) 594. - Phaeomeria imperialis Lindl. ex Ridl.,
Fl. Malay Penins. 4 (1924) 272; Elmer, Leafl. Phil. Bot. 8 (1915) 2907. Type:
Originally from Mauritius, 1825, C. Telfair s.n., cultivated (holo, K).

- Nicolaia intermedia Valeton, Bull. Jard. Bot. Buitenzorg ser. 3,3 (1921) 133.
Type: Possibly from Java, Anon. s.n. (HB XI B IV 101), cultivated in Bogor
(holo, BO).

Rhizome short-creeping. Leafy shoot to 6 m, several together in a loose
clump; base to 6 cm in diameter, sheath pubescent at the very base, +
reddish. Sheath green, striate when dry, glabrous, pruinose when young.
Ligule to 15 mm, entire, obtuse, green, glabrous, margin ciliate. Petiole to
25 mm. Lamina oblong, to 80 x 20 cm, yellowish or mid-green, pale green
beneath; average length to width ratio 3.5—6.5; base truncate, + unequal;
apex acuminate to c. 1 cm. Inflorescence (including peduncle) to 2 m, erect
from base of leafy shoot, receptacle elongate to 10 cm in old inflorescences,
with up to at least 320 flowers, 10-20 open at a time. Peduncle 0.6—2 m,
peduncular bracts to 22 x 2.5 cm (upper, which play the same role as the
lower sterile bracts), distichous, green to yellowish green, pubescent near
base. Spike to 15 x 15 cm, ovoid, flowers not extended above bracts. Sterile
bracts numerous, spirally arranged, lower to 13 x 4.5 cm, lowest one biggest,
distinctly reflexed, dark or pale pink with pale (sometimes almost white)
margin; turning brown with age, pubescent near base. Fertile bracts 2.5—7
x 0.7-2.5 cm, oblong, concave, apex obtuse, sometimes inrolled, red to pale
pink with a white margin, glabrous, apex with ciliate margin. Bracteole
2.2-3.1 cm, membranous, transparent, pale pink to red at apex, with two
fissures of 0.3-1.5 cm, glabrous, apex 2-toothed, tufted. Flower: Calyx 3-3.5
cm, reaching at least base of anther, pink, fissured 2 cm, glabrous; apex
irregularly 3-toothed, tufted. Corolla tube 2.7—-2.8 cm, dark red at apex,
sericeous ventrally below lobes, tube inside with two elongate, densely

Etlingera Giseke of Java 155

V-shaped hairy cushions 5 mm wide, c. 1 cm below labellum and coinciding
+ with attachment of the lobes on the outside, pubescent dorsally towards
filament. Lobes dark pink, glabrous with ciliate apex; dorsal lobe 21-23 x
5.5-6 mm, reaching slightly beyond stigma, narrowly elliptic to spathulate,
cucullate, apex obtuse, tufted; lateral lobes 22-24 x 3.5-4 mm, narrowly
elliptic to spathulate, cucullate, apex obtuse, slightly emarginate, with ciliate
tuft; attached straight, 2 mm below dorsal lobe. Staminal tube 10-12 mm;
labellum + entire, 20 x 18 mm, red with yellow margin, roughly papillose
in centre extending into the corolla tube, lateral lobes + erect, meeting
above stamen, margin slightly recurved, central lobe slightly emarginate,
extended c. 10 mm beyond anther, margins slightly recurved. Stamen 10-11
mm long; filament 2.5—-3 x 2.5 mm, white, papillose inside; anther 8-9 x 3.5
mm, + oblong, slightly wider at apex, erect, white to pale red, anther crest
short; thecae dehiscing in upper 1/2—2/3 almost to apex, pubescent, base with
long tuft. Style 3.7—3.8 cm, with scattered long hairs. Stigma 3 mm wide,
dark red, club-shaped; ostiole round, facing downwards. Ovary 4—-5.5 x 5
mm, sericeous; epigynous gland 4 mn, slightly bilobed, roughly papillose.
Infructescence remaining erect, head 10-12 (or longer) x 5—9 cm, the lower
bracts rot and are not conspicuous, 67-100 fruits per head; fruit 2.5-3.5 x
1.5-2.5 cm, pyriform, angled, top rounded, not beak-like, calyx + persistent,
to 3 mm, reddish, pink, pale orange-red or maroon, pubescent. Seeds to 4
mm across, rounded-angular. Plate IC.

Local names and uses: Blume (1827) listed the following names konje, hunje,
hunje-reuma (Sundanese). Heyne (1927) mentioned that rombe refers to
the young inflorescene and combrang (tjombrang orth. var.) to the flowers
(also Sundanese) and in Javanese or kecombrang, cumbrang, combrang.

Hondje hedjo (Sundanese; hedjo means green referring to the colour
of the leaves as opposed to E. hemisphaerica that has reddish leaves beneath;
Poulsen et al. 2293).

Edible leafy shoots, flowers, and fruits. Often sold at the market.

Etymology: The epithet refers to the raised inflorescence.

Ecology and habitat: It is difficult to say with certainty if Etlingera elatior is
native to Java as it has been cultivated there for a very long time. Bakhuizen
f. (1968) listed its occurence in primary and secondary forests to 1200 m,
though I am yet to encounter it in natural vegetation in Java.

Distribution: Widespread in the tropics (Poulsen, 2006).

Conservation status: LC (Least Concern). Not threatened.

156 Gard. Bull. Singapore 59 (1&2) 2007

Additional material examined: West Java Province, Gunung Kancana, 800
m, W of Parabuan village (6°54’S 107°03’E), 800 m, 26 Mar 2004, flowering,
Poulsen et al. 2293 (AAU, BO).

Notes: Etlingera elatior is most similar to E. hemisphaerica but the most
striking differences between the two is the the longer and reflexed lower
bracts in the involucre of E. e/atior. Furthermore, the inflorescence is usually
more than 1 m (vs. <1 m), the receptacle extends to 10 cm (vs. <2 cm), and
the fruit is reddish (not green).

The description by Bakhuizen f. (1968) mentions that the leaves can
be purple beneath which could resemble those of E. hemisphaerica. This I
have only observed in Java in cultivated plants. The colour of the bracts of
E. elatior can vary from deep blood-red to white (Poulsen, 2006). In Java,
some of the inflorescences sold in the market are deep red whereas the more
typical ones are pink.

In rare cases, such as the type of Diracodes javanica Blume, the
inflorescence in E. elatior appears terminally on a leafy shoot.

4. Etlingera foetens (Blume) R.M. Sm.

Etlingera foetens (Blume) R.M. Sm., Notes Roy. Bot. Gard. Edinburgh 43
(1986) 245; Poulsen, Etlingera of Borneo (2006) 132. Basionym: Elettaria
foetens Blume, Enum. Pl. Javae (1827) 52. Alpinia foetens (Blume) D. Dietr.,
Syn. Pl. 1 (1839) 12. Cardamomum foetens (Blume) Kuntze, Revis. Gen. PI.
2 (1891) 686. Amomum foetens (Blume) Benth. [Gen. Pl. 3 (1883) 644, nom.
inval.| ex Schumann, Bot. Jahrb. Syst. 27 (1899) 322; Valeton, Bull. Inst. Bot.
Buitenzorg 20 (1904) 44; Valeton, Icon. Bogor. 2 (1906) t. 157 figs. 16-17,
23, t. 154 fig. 18, t. 162 figs. 10-15; Bakhuizen f., Bekn. Fl. Java 18 (1958)
34; Bakhuizen f. in Backer, Fl. Java 3 (1968) 58. Achasma foetens (Blume)
Valeton, Bull. Inst. Bot. Buitenzorg 20 (1904) 93; Heyne, Nuttige pl. Nederl.
Indié 1 (1927) 489. Hornstedtia foetens (Blume) K. Schum., Pflanzenr. IV,
46 (1904) 200. Geanthus foetens (Blume) Valeton ex A.W.K. de Jong, Jaarb.
Dep. Landb. Nijverh. Handel Ned.-Indié 1917 (1919) 78. Type: C.L. von
Blume s.n., bar code L0193175 (holo L).

- Etlingera triorgyalis auct. non (Baker) R.M. Sm.; Etlingera triorgyalis vel
sp. aff.: Smith, Notes Roy. Bot. Gard Edinburgh 43 (1986) 443; Sakai & Nag-
amasu, Edb. J. Bot 60 (2003) 212.

Rhizome \ong-creeping (up to 80 cm between shoots), stout, c. 2 cm in
diameter, scales large (to 9 cm), cream, pale brown to reddish. Leafy shoot
2-5 m, leafless c. 1 m, with up to 22 leaves; base to 7.5 cm in diameter,

Etlingera Giseke of Java £57

reticulate with distinct and tufted cross bars, green or greenish brown.
Sheath mid- to yellow-green, roughly reticulate, + pubescent, margin
ciliate. Ligule 15-21 mm, entire, green, pubescent, margin densely tufted
ciliate at apex. Petiole to 25 mm, pubescent. Lamina narrowly obovate, to
83 x 14 cm, slightly plicate, green, glabrous above (rarely scabrid in centre
near base), pubescent beneath; average length to width ratio 4.5—-6; base
cuneate, + unequal; apex acuminate to 2 cm. Inflorescence (including
peduncle) (10—)18-—25 cm, with 18-24 flowers, 2-7 open at a time. Peduncle
(3—)9-13 cm, subterranean, peduncular bracts to 5 x 5 cm. Spike 6-9 x 3-5
cm, ovate-cylindrical, flowers extended 0-1 cm above the bracts, length
only including bracts: 8—9 cm. Sterile bracts 5—7, distichous, lower 4.5-5.5 x
4.5-5.5 cm, upper to 8.5 x 2.3-3.5 cm, ovate to broadly spathulate (upper),
rigid, mucronate, red, pubescent in lower third. Fertile bracts 6.5—8 x 0.7-1.5
cm, spathulate, membranous, translucent white with pinkish apex, densely
pubescent. Bracteole 5.4—6.5 cm, whitish with pink apex, with two fissures
of c. 1 and c. 1.5 cm, densely pubescent; apex bifid both lobes emarginate,
aristate 3 mm, densely hairy. Flower: Calyx 7—7.5 cm, reaching anther, +
as long as corolla lobes or slightly shorter, membranous, fissured 3—3.5 cm,
densely pubescent; apex irregularly 3-fid, aristate. Corolla tube 5.8—6.7 cm,
whitish with pink apex, glabrous or with scattered hairs, inside with 5 mm
band of scattered hairs c. 10 mm below labellum. Lobes pale red with bright
red apices, with a few hairs; dorsal lobe 20-22 x 6-7 mm, reaching middle or
apex of anther, spathulate, cucullate, margin constricted; lateral lobes 18-22
x 5-6 mm, spathulate, + cucullate; attached obliquely, 0-2 mm higher or at
same level as dorsal lobe. Staminal tube 11—12 mm; labellum 3-lobed, 38—43
x 17 mm, deep red with darker red and roughly papillose centre, glabrous,
lateral lobes erect, margins thin, involute over stamen, central lobe broadly
spathulate, 14-18 mm wide, + emarginate, apex extended 27-31 mm beyond
anther; anther subsessile, 10-11.5 x 2.5-5 mm, widest at apex, + erect, pale
pink or red, darker at crest; thecae dehiscing in upper 60%, few hairs at
the base, especially dorsally. Style 6.8—7.3 cm, hairy dorsally in upper part.
Stigma 4 mm, rounded-triangularly to pentangular with a rounded back,
pale red or red; ostiole transverse, 2 mm, facing downwards. Ovary 5-6 mm,
densely pubescent; epigynous glands 6.5 mm, bipartite, apices tooth-shaped.
Infructescence half embedded in the soil, head 4.5 x 3-8 cm, subglobose,
bracts shredding, to ca 22 fruits per head; fruit 2.2—3 x 2.5 cm, subglobular,
angled, with fine, papillose ridges, pink, densely pubescent. Seeds to 3 mm
across, rounded. Plate 1D.

Local names and uses: Tepus sigung (Sundanese). This name was recorded by
Blume (1827) and confirmed when Poulsen et al. 2296 was collected in 2004.
According to Valeton (1904), sigung (or sigeung) is the local name for the

158 Gard. Bull. Singapore 59 (1&2) 2007

Javan Skunk Marten (possibly Mydaeus javanensis) which is infamous for
its revolting smell, resembling asafoetida and fennel. The fruits are eaten.
Etymology: The epithet means smelly.

Ecology and habitat: Lowland primary or disturbed forests at banks of rivers
or streams to 950 m.

Distribution: Borneo, Java, Sumatra and possibly Peninsular Malaysia and
Thailand.

Conservation status: EN Blab(iii). Deforestation seriously threatens the
forest in Java — especially in the lowlands. Already Bakhuizen f. (1968)
noted that FE. foetens was rare and, during my surveys from 2003-2006, I
only found one sterile plant.

Additional material examined: West Java Province, Gunung Kancana, 2 km
WSW of Parabuan village (6 55’S 107 03’E), 950 m, 27 Mar 2004, sterile,
Poulsen et al. 2296 (AAU, BO).

Notes: Etlingera foetens is easily recognized by its deeply reticulate and
broad leaf bases, plain red flowers where the dorsal lobe of the corolla does
not cover the anther, the elongate and broad labellum and the strong smell
when crushed.

I have not seen flowering plants of EF. foetens in Java and thus the
description of floral characters are based on Bornean measurements, which,
however, match nicely those in the Flora of Java (Bakhuizen f., 1968). In
Borneo, all flowers observed so far are uniformly red whereas in Sumatra
the lateral margins of the labellum are occasionally slightly yellow. Thus it
would not be surprising if flowers in Java showed similar variation.

As discussed in detail by Poulsen (2006), E. triorgyalis (Baker) R.M.
Sm. is similar to E. foetens. It is sometimes a taller plant (8 m vs. 5 m) and
has larger sterile bracts (with recurved apices making the inflorescence
cyathiform), and a greater number of flowers per inflorescence. In
addition, several floral measurements (calyx, corolla, and width of apical
lobe of the labellum) are larger (Khaw, 2001). These characters seem
to separate the material from Peninsular Malaysia (including the type
of E. triorgyalis from Perak) from that of Java, Borneo and Sumatra.

5. Etlingera hemisphaerica (Blume) R.M. Sm.

Etlingera hemisphaerica (Blume) R.M.Sm.,Notes Roy. Bot. Gard. Edinburgh
43 (1986) 245. Basionym: Elettaria hemisphaerica Blume, Enum. pl. Javae
(1827) 51. Alpinia hemisphaerica (Blume) D. Dietr., Syn. pl. 1 (1839) 13.

Etlingera Giseke of Java 159

Nicolaia hemisphaerica (Blume) Horan., Prodr. Monogr. Scitam. (1862) 32;
Bakhuizen f., Bekn. Fl. Java 18 (1958) 46; Bakhuizen f. in Backer, Fl. Java 3
(1968) 63. Cardamomum hemisphaericum (Blume) Kuntze, Revis. gen. pl. 2
(1891) 686. Amomum hemisphaericum (Blume) K. Schum., Bot. Jahrb. Syst.
27 (1899) 307. Phaeomeria hemisphaerica (Blume) K. Schum., Pflanzenr. IV,
46 (1904) 263. Type: C.L. von Blume s.n. (holo, L), Java.

- Elettaria anthodioides Teijsm. & Binn., Ned. Kruidk. Arch. 3 (1855) 392.
Amomum anthodioides (Teijsm. & Binn.) Koord., Meded. Lands Plantentuin
19 (1898) 318. Phaeomeria anthodioides (Teijsm. & Binn.) Koord., Exkurs.-
Fl. Java 1 (1911) 332. Nicolaia anthodioides (Teijsm. & Binn.) Valeton, Bull.
Jard. Bot. Buitenzorg ser. 3, 3 (1921) 128. Type: J.E. Teijsmann s.n. (iso, P).

- Elettaria atropurpurea Teijsm. & Binn., Natuurk. Tijdschr. Ned.-Indié 24
(1862) 327. Phaeomeria atropurpurea (Teijsm. & Binn.) K.Schum., Pflanzenr.
IV, 46 (1904) 266. Nicolaia atropurpurea (Teijsm. & Binn.) Valeton, Bull.
Jard. Bot. Buitenzorg ser. 3, 3 (1921) 128; Heyne, Nuttige pl. Nederl. Indié 1
(1927) 487. Type: J.E. Teijsmann s.n.

- Nicolaia sanguinea Valeton, Bull. Inst. Bot. Buitenzorg 20 (1904) 36.
Phaeomeria sanguinea (Valeton) Koord., Exkurs.-Fl. Java 1 (1911) 332.
Type: Anon. s.n. (BO).

- Nicolaia rostrata Valeton, Bull. Jard. Bot. Buitenzorg ser. 3, 3 (1921) 134.
Phaeomeria rostrata (Valeton) Loes., Nat. Pflanzenfam. ed. 2, 15,A (1930)
594. Type: H.A.B. Biinnemeyer 809 (holo BO), Gunung Talamau (“Ophir’’)

- Nicolaia rostrata var. talangensis Valeton, Bull. Jard. Bot. Buitenzorg ser.
3,3 (1921) 135. Type: H.A.B. Biinnemeyer 5298, 5299, 5431 (syn, BO), West
Sumatra, Gunung Talang.

Rhizome in clump. Leafy shoot 3-6 m; base to 6-8 cm diam., bright red.
Sheath green or yelllow-green with reddish blotches, red when young,
glabrous. Ligule 12-13 mm, slightly emarginate, green. Petiole to 25 mm.
Lamina narrowly elliptic, to 80 x 15 cm, dark green, with pale green midrib
above; reddish or brownish beneath, margin undulating; average length to
width ratio 4.8—5.4; base cuneate to + auriculate. Inflorescence (including
peduncle) 18—81(—120) cm, erect, receptacle 12-15 mm, with 38-49 flowers,
1-4 open at a time. Peduncle to c. 1 m, peduncular bracts not completely
covering the green axis, uppermost enclosing spike, pale yellow-green. Spike
6-7 x 3-6 cm, cup-shaped, flowers only extending 5 mm above the bracts in
very mature inflorescences. Sterile bracts: 5, to 6 x 3.5 cm, ovate-elliptic, pale

160 Gard. Bull. Singapore 59 (1&2) 2007

pink tinged green at base ordarker red especially towards apex and with
a pale margin, glabrous. Fertile bracts 3-6 x 0.9-3.5 cm, cucullate, tinged
red, short-lived, glabrous (pubescent at the very base only). Bracteole to
2.5 cm, cream tinged red, with 2 fissures of 0.5—1.5 cm, apex bifid. Flower:
calyx c. 4 cm, reaching beyond apex of anther and shorter than corolla
lobes, red with yellow-green apex, fissured 2 cm, pubescent near base, apex
3-toothed, teeth close together. Corolla tube 2.5 cm, white, glabrous, tube
inside densely hairy from point of attachment of dorsal lobe on the outside
to base of anther and with distinct hairy cushions at point corresponding
to lateral lobe attachment. Lobes reaching beyond stigma, dark burgundy
red with white margin and apex; dorsal lobe 25 x 7 mm, cucullate, distinctly
rigid mucronate; lateral lobes 22 x 6 mm, attached oblique, converging, 0-3
mm above dorsal lobe. Staminal tube 12 mm. Labellum narrowly ovoid, 17
x 15 mm, dark red, yellowish white in centre, margin yellowish white, central
lobe extended 7.5 mm beyond anther. Stamen 11 mm long; filament 1.5 x 2
mm, cream; anther 9.5 x 3 mm, red; thecae dehiscing in upper half, margin
hairy. Style 3.5 cm, with scattered hairs. Stigma 2.5 mm wide, purple, ostiole
transverse elliptic, facing downwards. Ovary 3 x 3 mm; epigynous glands
3.5 mm, deeply bilobed, papillose. Infructescence to 12 x 10 cm, with 2-20
fruits per head, bracts not persistent; fruit yellowish green, pubescent, apex
truncate to slightly depressed. Mature infructescence with seeds not seen in
Java. Plate 1E.

Local names and uses: Hondje burem (Sundanese; referring to the leaves
being red beneath; Poulsen et al. 2295). Hunje leaweung (Sundanese; Blume,
1827 — according to Valeton (1904) leuweung means wood; he considered
E. hemisphaerica the wild origin of E. elatior). Heyne (1927) added the
names hondje hedjo and hondje laka (based on what he called Nicolaia
atropurpurea); these local names are also given for E. elatior and E. solaris,
respectively.

Leafy shoot and fruit edible. According to Bakhuizen f. (1968),
cultivated locally.

Etymology: The epithet means hemispherical, probably referring to the cup-
shaped inflorescence.

Ecology and habitat: Primary and secondary lowland forests to 950 m. Fruits
emptied by rodents.

Distribution: Sumatra, Java, and probably Peninsular Malaysia and Thailand.
There are no definite records of this species from the wild in Borneo but it
is cultivated at Tenom Agricultural Park in Sabah. A. Lamb (pers. comm.)

Etlingera Giseke of Java 161

found it near Tenon and believes it was introduced by Javanese workers
who came to work in the tobacco estates at about 1850 and took useful
plants with them from Java.

Conservation status: VU Blab(iii). Vulnerable by extent of occurrence
estimated <20,000 km , known from <10 locations, and decline in the extent
and quality of lowland forest habitats in Java.

Additional materials examined: Banten Province: buffer zone of Ujong
Kulon NP, Cikacang (6 48’S 105 32’E), 130 m, 28 Apr 2005, fruiting, Poulsen
et al. 2347 (BO, E). West Java Province: Gunung Kancana, 2 km WSW of
Parabuan village (6 55’S 107 03’E), 950 m, 27 Mar 2004, sterile, Poulsen et
al. 2295 (AAU, BO); Gunung Tutupan, (7°22’S 106 42’E), 150 m, 11 Aug
2006, flowering and fruiting, Poulsen et al. 2460 (AAU, BO, E, L).

Notes: Amongst the Etlingera presently known in Java, E. hemisphaerica
is most similar to E. elatior from which it differs in its erect bracts (not
reflexed) and in having a lamina that is reddish beneath. Floral differences
between the two species seem to be minor except for the anther possibly
being longer in EF. hemisphaerica, but more material is needed to test this.

With its leaves being wine-red beneath, Etlingera pyramidosphaera
in Borneo appears very similar to E. hemisphaerica but the former differs
in having a narrower inflorescence with fewer flowers, the anther thecae
dehiscing for their entire length, and in its beaked fruits.

Bakhuizen f. (1968) described the fruits as globular or spindle-shaped,
beaked. In the revision of Etlingera of Borneo, Poulsen (2006) emphasized
the fruit shape as a reliable character. Thus, in the present account above, the
fruit is described as globular — not beaked! More material will be necessary
to establish if beaked fruits of hemisphaerica-like plants actually occur in
Java, and if these plants deserve taxonomic recognition.

6. Etlingera megalocheilos (Griff.) A.D. Poulsen

Etlingera megalocheilos (Griff.) A.D. Poulsen, Etlingera of Borneo (2006)
167. Basionym: Achasma megalocheilos Griff., Not. Pl. Asiat. 3 (1851) 426,
Ic. Pl. Asiat. 3 (1851) t. 355; Valeton, Icon. Bogor. 2 (1906) t. 188, t. 199 figs.
7-9; Heyne, Nuttige pl. Nederl. Indié 1 (1927) 489; Holttum, Gard. Bull.
Singapore 13 (1950) 191, fig. 23; Burtt & Smith, Notes Roy. Bot. Gard.
Edinburgh 31 (1972) 221, fig. 16. Amomum megalocheilos (Griff.) Baker,
Fl. Brit. India 6 (1892) 236; Valeton, Bull. Inst. Bot. Buitenzorg 20 (1904)
44; Valeton, Icon. Bogor. 2 (1906) t. 188 figs. 1-8, t. 199 figs. 7-9; Bakhuizen
f., Bekn. Fl. Java 18 (1958) 33; Bakhuizen f. in Backer, Fl. Java 3 (1968) 57.
Hornstedtia megalocheilos (Griff.) Ridl., J. Straits Branch Roy. Asiat. Soc.

162 Gard. Bull. Singapore 59 (1&2) 2007

32 (1899) 146; Schumann, Pflanzenr. IV, 46 (1904) 199; Ridley, Matr. Fl. Mal.
Penin. 2 (1907) 38; Ridley, Fl. Mal. Penin. 4 (1924) 270. Type: W. Griffith s.n.
(specimen not found). Malaysia, Johore, Mt. Ophir, flowering in February.

- Amomum rubroluteum Baker, Fl. Brit. India 6 (1892) 236. Etlingera
rubrolutea (Baker) C.K. Lim, Folia Malaysiana 2(3) (2001) 157. Type: A.C.
Maingay 1588 (holo, K), Malaysia, Malacca.

- Etlingera littoralis auct. non (J. Konig) Giseke: Smith, Notes Roy. Bot. Gard
Edinburgh 43 (1986) 445; Lim, Folia Malaysiana 2 (2001) 149; Keng et al.,
Concise Fl. Singapore 2 (1998) 57; Khaw, Gard. Bull. Singapore 53 (2001)
235!

Rhizome long-creeping, subterranean (1.5—25 cm), stout, >2 cm in diameter,
cream to pale brown, scales to 6 cm, brown, pubescent at base. Leafy shoot
to 5 m, with up to 28 leaves; base to 8 cm in diameter, dark green. Sheath
striate with some cross bars, especially in upper part of the shoot, glabrous,
green when fresh. Ligule to 35 mm, entire, green or tinged reddish brown,
glabrous or with a few scattered hairs, margin ciliate. Petiole 25-55 mm,
glabrous. Lamina to 101 x 16 cm, oblong, broadest above the middle, mid-
to dark green, pale beneath, young leaf tinged reddish, glabrous (rarely
pubescent); average length to width ratio 3.5—7; base + unequal; apex acute.
Inflorescence (including peduncle) 9-18 cm, embedded in the soil, often
some distance from base of leafy shoot, with 10-12 flowers, 2-5 open at a
time. Peduncle 2-10 cm, subterranean, peduncular bracts cream, acute, shiny,
glabrous. Spike to 10-12 x 2-3 cm, cylindrical, flowers extended 3-4 cm above
the bracts, length only including bracts: 5—8 cm. Sterile bracts c. 5, loosely
and spirally arranged, to 4—7 x 1.5-3.5 cm (upper longest and narrowest),
ovate to broadly spathulate (widest above the middle), rigid, mucronate,
cream tinged pink or bright red, densely pubescent at least in lower half.
Fertile bracts 5—8.5 x 0.6—1.9 cm, linear to spathulate, semitransparent, white,
pubescent in lower half; apex cucullate, ciliate. Bracteole 4.5—7 cm, pale pink,
membranous, with two fissures of 1.5—2.5 cm, pubescent in lower half, apex
2-toothed, ciliate. Flower: Calyx 6.1—-9 cm, almost reaching filament, + as
long as corolla lobes, white to pale red with pinkish apices, fissured 2.5—3.5
cm, pubescent in lower 1/4; apex irregularly 3-toothed, tufted. Corolla tube
5.8-8 cm, pale red, darker at apex, glabrous, tube hairy inside especially in a
10 mm band ending 10 mm from labellum. Lobes pale red or pink, glabrous,
delicately membranous; dorsal lobe 21-30 x 7-9 mm, reaching near middle
of anther (but pushed to the side by the lateral lobes of labellum leaving
the anther + exposed), elliptic, broadest below middle, apex slightly ciliate;
lateral lobes 21-25 x 4.5—5 mm, linear-elliptic, broadest below middle, apex

Etlingera Giseke of Java 163

slightly ciliate; insertion oblique, converging, 0-3 mm above dorsal lobe.
Staminal tube 12-22 mm; labellum hourglass-shaped, 52-70 x 20-22 mm,
plain red or red to orange-red with yellow margin, with a longitudinal
central ridge, glabrous, lateral lobes erect, adhering to sides of anther, base
slightly auriculate, central lobe 40-48 x 17 (measured from apex of anther
and when flattened), spathulate, entire or emarginate (to 1.5 mm), margin
recurved, apex extended 35 mm beyond anther. Stamen 17 mm; filament
4-7 x 4-5 mm, slightly hairy on outside, pale red; anther 10-11.5 x 5—5.5
mm, broadest at apex, emarginate 1.5-2.5 mm, slightly angled 135-160 , red,
darker at crest; thecae dehiscing in upper 1/2—2/3, glabrous with a few hairs
at the base. Style 8.5—9.5 cm, glabrous to very sparsely hairy adaxially near
apex. Stigma 3-4 mm wide, rounded-triangular with a rounded back, pale
or dark red; ostiole transverse, 2.5—3 mm, facing downwards or forwards,
perhaps flexistylous. Ovary 3-6 x 3-4 mm, densely hairy; epigynous gland
5~9 mm, deeply bilobed or bipartite, apex sometimes hairy. Infructescence
embedded in the soil, head ca 5 x 7-8 cm, bracts not persistent; fruit 2.5—3.5
cm across, rounded, not ridged, sometimes slightly warty at apex, pale brown
or pink, densely pubescent. Seeds up to 4 mm across, angular. Plate 1F.

Local names and uses: Tepus (Sundanese; Heyne, 1927). The smell is variable;
at least in Borneo (Poulsen, 2006) and in Sumatra the smell is strong and
somewhat unpleasant, similar to E. foetens.

According to Heyne (1927), E. megalocheilos was not cultivated but
the fruits searched for in the wild and eaten.

Etymology: The epithet refers to the large labellum.

Ecology and habitat: Often dominant in forest gaps or completely open
areas to 1300 m.

Distribution: Malay Peninsula, Singapore, Sumatra, Java, and Borneo.

Conservation status: Least concern (LC). Bakhuizen f. (1968) thought it
scarce everywhere, but I have observed it in several very open habitats and
consider it rather resilient to disturbance. It may actually have expanded in
recent years.

Additional materials examined: Banten Province, Ujung Kulon NP, Cibayoni
(6 41’S 105 35’E), 100 m, 26 Apr 2005, flowering, Poulsen et al. 2341 (AAU,
BO, E). West Java Province, Cibabi (7 18’S 106 24’E), 50 m, 12 Aug 2006,
flowering, Poulsen et al. 246] (BO, E).

164 Gard. Bull. Singapore 59 (1&2) 2007

Notes: Etlingera megalocheilos is most easily confused with E. coccinea that
also has the inflorescence embedded in the soil and an elongate, red and
yellow labellum. But in EL. megalocheilos the anther is not covered by the
corolla lobe, the margins of the labellum are not inrolled, and the labellum is
red with more or less pale red or yellowish lateral lobe margins (not yellow
with red margins).

Griffith (1851) described Achasma megalocheilos from Peninsular
Malaysia — a taxon also mentioned by Ridley (1899), Holttum (1950), and
which Khaw (2001) called E. littoralis following Burtt and Smith (1986).

I have not encountered fruits of E. megalocheilos in Java but those
I have seen from Borneo (Poulsen, 2006) and Sumatra and also described
by Holttum (1950) match Valeton’s (1906) description and illustrations of
the fruits of A. megalocheilos which are rounded and smooth but with a few
warty protuberances near the top, based on material from Malabar Mts.,
Java.

7. Etlingera solaris (Blume) R.M. Sm.

Etlingera solaris (Blume) R.M. Sm., Notes Roy. Bot. Gard. Edinburgh 43
(1986) 249; PROSEA 13 (1999) 254. Basionym: Elettaria solaris Blume,
Enum. pl. Javae (1827) 52. Alpinia solaris (Blume) D. Dietr., Syn. pl. 1 (1839)
12. Nicolaia solaris (Blume) Horan., Prodr. Monogr. Scitam. (1862) 32;
Heyne, Nuttige pl. Nederl. Indié 1 (1927) 488; Bakhuizen f., Bekn. Fl. Java 18
(1958) 45; Bakhuizen f. in Backer, Fl. Java 3 (1968) 63. Cardamomum solare
(Blume) Kuntze, Revis. gen. pl. 2 (1891) 687. Amomum solare (Blume) K.
Schum., Bot. Jahrb. Syst. 27 (1899) 308; Valeton, Icon. Bogor. 2 (1906) t. 160
figs. 15, 13-15, t. 165 figs. 6-12, 16; Phaeomeria solaris (Blume) K. Schum..,
Pflanzenr. IV, 46 (1904) 264. Type: C.L. von Blume s.n. (L).

- Elettaria pallida Blume, Enum. pl. Javae (1827) 52. Alpinia pallida (Blume)
D. Dietr., Syn. pl. 1 (1839) 12. Nicolaia pallida (Blume) Horan., Prodr.
Monogr. Scitam. (1862) 32. Cardamomum pallidum (Blume) Kuntze, Revis.
gen. pl. 2 (1891) 687. Phaeomeria pallida (Blume) K. Schum., Pflanzenr. IV,
46 (1904) 264. Type: C.L. von Blume s.n. (holo L), Java.

Rhizome short-creeping (10-20 cm between closest pairs of leafy shoots),
4 cm diameter, scales dehiscent, brownish, papery. Leafy shoot 5 m; base
to 5-7 cm diameter, brownish. Sheath: lower caducous, brownish; upper
yellowish green with pubescent reticulation. Ligule 40-80 mm, membranous,
caducous, deeply bilobed. Petiole 15-20 mm. Lamina to 83 x 19 cm, narrowly
elliptic or obovate, slightly plicate, dull mid-green, midrib yellow-green, pale
green with yellow-green midrib beneath, glabrous; average length to width
ratio 3.25—5.25; base + unequal, cuneate. Inflorescence (including peduncle)

Etlingera Giseke of Java 165

to 50 cm, prostrate or ascending to erect, receptacle 3-8 cm (longest in
infructescence), with numerous flowers, ca 10 open at a time. Peduncle to 40
cm, peduncular bracts, upper as long as lowest sterile bracts, to 9 x 3-4 cm.
Spike 11 x 12 cm, globose, robust, flowers not extending above the bracts.
Sterile bracts: lower 7-8 x 2-4 cm, with membranous margin and conspicuous
apex (to 25 mm with inrolled margin, horn-like twisted towards centre of
inflorescence), red soon turning brown, densely pubescent especially at base
and margin. Fertile bracts to 5—7 x 1-1.2 cm, similar in shape to sterile bracts,
orange-red. Bracteole 4—6 cm, with red apex, one long fissure to 5-15 mm
above base; sometimes a second fissure for 5 mm only, densely pubescent
throughout, apex bifid with 2 mucro (thus sometimes appearing 3-toothed).
Flower: calyx 4.5—6 cm, reaching to apex of anther and beyond corolla lobes,
red, fissured 3 cm, pubescent, apex 3-toothed 5-9 mm. Corolla tube 3-4
cm, + pubescent in lower half, tube inside with an opposite V-shaped hairy
cushion coinciding with dorsal corolla lobe attachment and a V-shaped one
coinciding with the lateral lobes on the outside, ca 22 mm below labellum.
Lobes orange-red, with scattered hairs; dorsal lobe 25-26 x 3-3.5 mm,
linear, apex acute, reaching to base of anther; lateral lobes 23 x 2-3 mm,
linear, insertion oblique, diverging, 3 mm below dorsal lobe. Staminal tube
17-22 mm. Labellum rounded triangular to ovate, 20-23 x 20 mm, orange-
red; lateral lobes, margin yellow, central lobe extended 4-8 mm beyond
anther, margin curved outwards. Stamen 16 mm: filament 2-3 mm x 3-3.5
mm, white to pale red; anther 13-14 x 3.5 mm, linear, + erect, red, anther
crest bilobed; thecae dehiscent in upper half 4-5 mm to 2 mm below apex,
pubescent. Style 5—5.5 cm, with scattered hairs. Stigma 3—3.5 mm wide, dark
purple, heart-shaped with scattered hairs; ostiole transverse, facing down- or
forwards (possibly flexistylous); ovary 5-8 x 5 mm, pubescent. Epigynous
glands 44.5 mm, with one incision, apex irregular, bilobed, margin curved
inwards. Infructescence lying on ground (because of the heavy fruits), head
20 x 20-25 cm, globose, bracts persistent (at least the bases); fruit 10 x 4 cm,
angularly obovoid, beaked, (broadest ca 4 cm from base), 3- to 6-sided, with
persistent calyx, red and juicy when ripe, pubescent. Seeds 4 mm diameter,
rounded. Plate 1G.

Local names and uses: Hondje warak (Sundanese: Blume, 1827; Poulsen et
al. 2297), honje laka, honje ngoser (Heyne, 1927). Fruit edible.

Etymology: The epithet means sun-like probably referring to the
inflorescence at anthesis. In the Mountain Flora of Java, van Steenis (1972)

mentions E. solaris as earth sun”.

Ecology and habitat: Montane forests near streams at 800-1750 m.

166 Gard. Bull. Singapore 59 (1&2) 2007

Distribution: Sumatra, W Java as far east as Gunung Merapi in Central Java.

Conservation status: VU Blab(iii). Vulnerable by extent of occurrence
estimated <20,000 km of montane forest, known from <10 locations, and
decline in extent and quality of habitat.

Additional materials examined: West Java Province: Halimun NP, Citalahab
(6'44’S 106°31’E), 1100 m, 21 Mar 2004, flowering and fruiting, Poulsen et al.
2285 (AAU, BO, E, L); Gede-Pangrango NP, Cibodas (6°45’S 107°59’E), 1750
m, 28 Mar 2004, flowering and fruiting, Poulsen et al. 2297 (AAU, BO).

Notes: The peduncle of Etlingera solaris is variable in position and direction
but the species 1s easily recognized by the long and deeply bilobed ligule and
the horn-shaped, twisted, pubescent bracts. Valeton (1921a, p. 137, plate 6)
described FE. solaris var. aurantiaca from Gunung Salak, Java. The variety is
supposed to have an erect inflorescence to 20 cm (not procumbent), shorter
teeth to the calyx and the lip orange rather than dark red, and possibly the
same as Elettaria pallida Blume. I have seen this at Halimun and agree with
Bakhuizen f. (1968) that it is hardly different. A collection (Poulsen 2418)
from Gunung Kerinci, Sumatra, had its inflorescence embedded in the
ground as the peduncle was subterranean, the stamen was shorter (13-14
mm) but the anther dehiscence matched that in the Javanese material.

Amomum chrysocalyx K. Schum. was listed as a synonym by
Bakhuizen f. (1968) but, after inspecting its type, I am convinced that it has
has no relevance to E. solaris.

Incompletely Known Species

8. Etlingera heyniana (Valeton) R.M. Sm.

Etlingera heyniana (Valeton) R.M. Sm., Notes Roy. Bot. Gard. Edinburgh
43 (1986) 246. Basionym: Nicolaia heyniana Valeton, Bull. Jard. Bot.
Buitenzorg ser. 3, 3 (1921) 132; Heyne, Nuttige pl. Nederl. Indié 1 (1927)
488. Phaeomeria heyniana (Valeton) Burkill, Bull. Misc. Inform. (1935)
318. Amomum heynianum (Valeton) Bakhuizen f., Bekn. Fl. Java 18 (1958)
31; Bakhuizen f. in Backer, Fl. Java 3 (1968) 56. - Type: Sentiong, Jakarta,
cemetery, possibly cultivated, Nov 1918, K. Heyne s.n. (holo, BO).

No recent material has been seen and the modified description below is
only a summary of what is presented by Valeton (1921a) and Bakhuizen f.
(1968).

Leafy shootto 4m. Sheath glabrous. Ligule ca 8 mm, elliptic, obtuse, glabrous,
stout. Petiole to 5 mm. Lamina 19-28(-65) x 4.5-5.5(-17) cm, narrowly

Etlingera Giseke of Java 167

obovate, glabrous throughout; length to width ratio 4-6; base acute; apex
shortly caudate-acuminate; margin glabrous. Inflorescence fusiform, red:
receptacle discoid, with <20 flowers (estimated from Valeton, 1921a, Plate
3), ca 4 open at a time. Peduncle short, curved, scales obovate, glabrous, red,
apex rounded, mucronate. Spike + ovoid, only including bracts: to 5.5 x 0.5-1
cm. Sterile bracts 5.5—-6.5 x 3 cm, oblong. Fertile bracts to 2—5.5 x 0.5-1 cm.
Bracteole c. 4 cm, glabrous. Flower: calyx 5 cm, glabrous, 3-toothed. Corolla
lobes 25 x 5 mm, linear. Labellum 3 x 1.5 cm long, with about equal elliptic
upper and lower halves separated by a distinct constriction, red with yellow
margin; central lobe ca 10 mm wide, margin slightly curled. Filament 7 mm;
anther ca 10 mm, crest bilobed, divergent. Style glabrous. Stigma discoid,
ostiole transverse; ovary pubescent. Epigynous glands 4 mm. Infructescence
unknown.

Local names and uses: Hondje (Heyne, 1927). Valeton (1921a) noted that
the entire plant is strongly aromatic like Nicolaia speciosa (E. elatior).

Etymology: The epithet is in honour of the Dutch botanist Karel Heyne
(1877-1947) who collected in Java and Sumatra.

Ecology and habitat: Unknown. Seems to tolerate growing in a rather open
habitat.

Distribution: Java.
Conservation status: Unknown.

Notes: The illustration of Etlingera heyniana in Valeton (1921a, Plate 3) looks
to me more like an inflorescence of FE. megalocheilos where the flowers
have not fully opened yet, similar to what may be observed in E. nasuta
(K. Schum.) R.M. Sm. in Borneo. Apart for the labellum being erect and
significantly shorter in E. heyniana, there are not stron evidence to separate
them. At least the colours of the labellum (red with yellow margin) are the
same. The type at BO mentions Gunung Honje as the locality— not Sentiong.
I thus went to one G. Honje, of which locality there might actually be several
in Java. At the locality I visited near Ujung Kulon, EF. megalocheilos was
very common. A closer study of the floral development of this species may
be fruitful.

Even though Valeton (1921a) mention the distinct smell of E.
heyniana, this is not strong evidence against the possibility of synonymy with
E. megalocheilos, as in Sumatra I have experienced that it may sometimes

168 Gard. Bull. Singapore 59 (1&2) 2007

have a strong smell.

9. Etlingera walang (Blume) R.M. Sm.

Etlingera walang (Blume) R.M. Sm., Notes Roy. Bot. Gard. Edinburgh 43
(1986) 251; PROSEA 13 (1999) 254. Basionym: Donacodes walang Blume,
Enum. pl. Javae (1827) 55. Alpinia walang (Blume) D. Dietr., Syn. pl. 1 (1839)
13. Elettaria walang (Blume) Mig., Fl. Ned. Ind. 3 (1859) 603. Cardamomum
walang (Blume) Kuntze, Revis. gen. pl.2 (1891) 687. Achasma walang (Blume)
Valeton, Bull. Inst. Bot. Buitenzorg 20 (1904) 93. Amomum walang (Blume)
Valeton, Bull. Inst. Bot. Buitenzorg 20 (1904) 44; Valeton, Icon. Bogor. 2
(1906) t. 162 figs. 1-9; Bakhuizen f., Bekn. Fl. Java 18 (1958) 32; Bakhuizen f.
in Backer, FI. Java 3 (1968) 56. Hornstedtia walang (Blume) Koord.-Schum.,
Syst. Verz. 1 (1911) 21. Type: Java, C.L. von Blume s.n. (holo, L).

- Elettaria glaberrima Zoll. & Moritzi, Syst. Verz. (1845-1846) 84.
Cardamomum glaberrimum (Zoll. & Moritzi) Kuntze, Revis. gen. pl. 2
(1891) 686. Type: H. Zollinger 670 (isotypes, BM, K, L, P).

Norecent material has been seen and the description below is only asummary
of what is presented by Valeton (1904; 1906) and Bakhuizen f. (1968).

Rhizome slender. Leafy shoot 1.5—2 m. Sheath glabrous. Ligule c. 10 mm,
finely ciliate, otherwise glabrous, stout. Petiole 5-10 mm. Lamina 29-49 x
5—6.5 cm, narrowly obovate, glabrous throughout; average length to width
ratio 6—7.5; base + unequal, narrowly cuneate; apex shortly acuminate,
finely hairy; margin glabrous. Inflorescence: receptacle almost flat, with <20
flowers, c. 3 open at a time. Peduncle to 8 cm, subterranean, scales to 5 x 1.3
cm, mucronate, finely longitudinally veined. Spike ovoid-cylindrical, only
including bracts: to 7 x 2.5-3.5 cm; about 5 sterile bracts, narrowly obovate,
acuminate, mucronate: to 6-8 x 1.3 cm. Fertile bracts to 6 cm, narrower than
sterile bracts. Bracteole c. 5 cm, bifid, densely pubescent at least in lower
half. Flower: calyx as long as corolla, irregularly 3-toothed. Corolla red; lobes
erect, oblong, dorsal lobe longest. Labellum yellow with red margin; lateral
lobes, margin curved upwards and conspicuously crenate, central lobe 5 cm
long, narrow, ligulate-spathulate, deeply bilobed. Filament 7 mm; anther
ca 10 mm, crest bilobed, divergent. Style 5.5 cm, hairy below apex. Stigma
triangular, hairy; ovary pubescent. Epigynous glands 4 mm. Infructescence
unknown.

Local names and uses: Walang (Sundanese). Leaves are served as a side dish
with rice (Valeton, 1904), as a condiment, or the leaves are burnt on rice
fields as an insect repellent (Heyne, 1927).

Etlingera Giseke of Java 169

Etymology: The epithet refers to the bad-smelling rice bug, walang sangit
(Leptocorisa acuta Thunb. or L. varicornis Fabr.). Bakhuizen f. (1968) noted
that all parts — especially the leaves — are ill-smelling.

Ecology and habitat: forests to 1200 m, and cultivated.
Distribution: Java.
Conservation status: Impossible to assess.

Notes: In Blume’s protologue (1827) of Etlingera walang he placed a question
mark after the genus (Donacodes). All that is reported of the new species is
that the leaves are elongate-linear-lanceolate, acuminate, and glabrous, and
it appears he had not seen the flowers. I think the question mark refers to
the uncertainty of which genus to place it in, but, without the flowers, one
has to wonder why Blume placed it in Donacodes (the remaining species of
which are presently placed in Hornstedtia).

Valeton (1904) did detailed studies around Bogor where he found
that EF. walang was ‘one of the most economically important Sundanese
plants’ and often cultivated, of unknown origin and very easily recognized
by its characteristic smell that stays for months with the specimen after
drying. He studied the inflorescence in detail (see plate in Valeton, 1906) and
thought it was much more narrow than that of E. coccinea and E. foetens but
that these three species formed a natural group and apart from the crenate
margin to the labellum, FE. walang was very similar to E. coccinea.

In my opinion what Valeton illustrated is just an E. coccinea with a
deeply bifid apex to the labellum, similar to Poulsen et al. 2343 from Ujung
Kulon. It is important to remember, however, that the basis for Valeton’s
descriptions (1904; 1906) and illustrations (1906; Plate 162, figs. 1-9) are not
based on the type.

The issue of smell is, however, very interesting. As mentioned in the
notes above on E. coccinea, in Borneo a very smelly form of this species
— also of unknown origin — is often cultivated and commonly sold in the
markets.

More detailed surveys around Bogor may result in the discovery
of walang and it would then be possible to establish if this just a form of E.
coccinea.

Conservation of Etlingera in Java

None of the seven well-known species of Etlingera is endemic to Java. They
all display geographical affinity with nearby Borneo or Sumatra to the north
and west, but no overlap with species in Wallacea to the east.

170 Gard. Bull. Singapore 59 (1&2) 2007

Plate 1. Photographs of Javanese Etlingera species. A. E. brachychila. Ujung Kulon, Banten
Province (Funakoshi IU 19) by H. Funakoshi; B. E. coccinea. Cikaso, W Java Province
(Poulsen et al.2458) by A.D. Poulsen; C. E. elatior. Batang Gadis NP, North Sumatra Province
(not collected) by Lis Maidi Darjo; D. E. foetens. Bau, Sarawak, Borneo (Poulsen et al. 1894)
by A.D. Poulsen; E. E. hemisphaerica. W Java Province (Poulsen et al. 2460) by A.D. Poulsen;
F. E. megalocheilos. Baduy Territory, Banten Province (not collected) by M. van Heist; G. E.
solaris. Halimun NP, W Java Province (Poulsen et al. 2285) by A.D. Poulsen.

Etlingera Giseke of Java 172

Two of the species (E. coccinea and E. megalocheilos) seem to be
common in very disturbed habitats and there is no great concern for their
conservation. However, there is reason to fear the future survival in Java of E.
brachychila, E. foetens, E. hemisphaerica, and to a lesser extent, of E. solaris.

Acknowledgements

I thank the Indonesian Institute of Sciences (LIPI) for permission to conduct
research in Java; the Herbarium Bogoriense and its Directors and staff for
logistic and other support, especially Marlina Ardiyani, Deden Girmansyah,
Ismail Rachman, Agus Sayadi, Aden Muhidin, and Alex Sumadijaya; The
National Parks Service of Halimun, Gede-Panggrango, and Ujong Kulon;
the local people in W Java for assistance in the field; H. Funakoshi for sharing
his important material of Etlingera brachychila with me; The National Parks
of Singapore for funding my journey to Asia in 2006; The Royal Botanic
Garden Edinburgh for providing facilities and contributing to the fieldwork
in Java in 2006; Mark F. Newman for giving constructive comments on an
earlier stage of this paper; Hidetoshi Funakoshi, Lis Maidi and Miriam
van Heist for letting me use their images; and the Carlsberg Foundation,
Denmark, for a senior research stipend.

References

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Bakhuizen van den Brink, R.C. 1968. Zingiberaceae, pp. 41-76. In: C.A.
Backer & R.C. Bakhuizen van den Brink, Flora of Java, vol. 3. N.V.P.
Noordhoff, Groningen, The Netherlands, 761 pp.

Blume, C.L. 1827. Enumeratio Plantarum Javae et insularum adjacentium.
J.W. van Leenwen.

Burtt, B.L. and Smith, R.M. 1986. Etlingera: the inclusive name for Achasma,
Geanthus and Nicolaia (Zingiberaceae). Notes from the Royal Botanic
Garden Edinburgh 43: 235-241.

Griffith, W. 1851. Notulae ad plantas Asiaticas, vol. 3. Calcutta.
Heyne, K. 1927. Zingiberaceae, pp. 473-504. In: De nuttige planten van

Nederlandsch Indié 1.2” edition. Department van Landbouw, Nijverheid
en handel in Nederlandsch Indié.

172 Gard. Bull. Singapore 59 (1&2) 2007

Holttum, R.E.1950.The Zingiberaceae of the Malay Peninsula. The Gardens’
Bulletin Singapore 13: 1-249.

IUCN 2000. JUCN Red List Categories and Criteria. Version 3.1.

Kato, M., Itino, T and Nagamitsu, T. 1993. Melittophily and ornithophily of
long-tubed flowers in Zingiberaceae and Gesneriaceae in West Sumatra.
Tropics 2: 129-142.

Khaw, S.H.2001.The genus Etlingera (Zingiberaceae) in Peninsular Malaysia
including a new species. Gardens’ Bulletin Singapore 53: 191-239.

Poulsen, A.D. 2006. Etlingera of Borneo. Natural History Publications
(Borneo), Kota Kinabalu, Malaysia & Royal Botanic Garden Edinburgh,
Scotland, 263 pp.

Ridley, H.N. 1899. The Scitamineae of the Malay Peninsula. Journal of the
Straits Branch of the Royal Asiatic Society 32: 85-184.

Sakai, S., Kato, M. & Inoue, T. 1999. Three pollination guilds and variation in
floral characteristics of Bornean gingers (Zingiberaceae and Costaceae).

American Journal of Botany 86: 646-658.

Smith, R.M. 1986. A review of Bornean Zingiberaceae: II (Alpineae, con-
cluded). Notes from the Royal Botanic Garden Edinburgh 43: 439-466.

Schumann, K.M. 1904. Zingiberaceae. In: A. Engler, Das Pflanzenreich IV,
46: 1-458.

Valeton, T. 1904. Ueber neue und unvollstandig bekannte Zingiberaceae aus
West-Java und Buitenzorg. Bulletin de Institut Botanique de Buitenzorg
20: 1-99.

Valeton, T. 1906. [cones Bogoriensis, vol.2. Leiden.

Valeton, T. 1921a. Nicolaia Horan. Description of new and interesting species.
Bulletin Jardin Botanique Buitenzorg 3: 128-140.

Valeton, T. 1921b. Rhynchanthus Hook. f., Geanthus Reinw. and Geostachys
Ridl. Bulletin Jardin Botanique Buitenzorg 3: 141-147.

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Gardens’ Bulletin Singapore 59 (1 &2): 173-182. 2007 173

Planting Date and Night Break Treatment Affected
Off-Season Flowering in Curcuma alismatifolia Gagnep.

S. RUAMRUNGSRI "’, J. UTHAI-BUTRA °, O. WICHAILUX *
AND P.APAVATJRUT ”

‘ Department of Horticulture, Faculty of Agriculture, Chiang Mai University,

Chiang Mai 50200, Thailand.

* HM. the King’s Initiative Centre for Flower and Fruit Propagation,

Chiang Mai University, Chiang Mai 50200, Thailand

* Department of Biology, Faculty of Science, Chiang Mai University,

Chiang Mai 50200, Thailand
* Department of Agricultural Extension, Ministry of Agriculture,
Bangkok, Thailand.

Abstract

Off-season flowering of Curcuma alismatifolia Gagnep. was studied in
Chiang Mai Province of Thailand where the weather in winter is cool with
temperatures between 16 to 30 "C, RH from 65 to 70 %, and 10 hrs of daylight.
Rhizomes were stored at 15 C for the 6 months from February to July,
2004. After root emergence, plants were grown under different night break
treatments. Night break treatments were conducted by supplying 2 hrs of
light daily from 20.00 to 22.00 hrs. The light source was 100 watt incandescent
light bulbs. There were three treatments: T1, night breaks supplied from
sprouting of the first shoot until the floral spike reached one inch long; T2 as
T1, but continued until the first floret opened; T3, was a control treatment
with no night break. Each treatment was carried out at different planting
dates, 1.e., August 9, September 9, October 9 and November 9. Plant height,
number of plants per cluster, flowering percentage and flower qualities
(number of coma bracts, number of green bracts, spike length and length
of flower stalk) were recorded. The results showed that plant growth and
flower qualities were similar with and without the night break treatment at
the 9 August planting date. However, the September to October planting
dates required night break treatments to promote flowering and maintain
flower qualities.

174 Gard. Bull. Singapore 59 (1&2) 2007

Introduction

Curcuma alismatifolia Gagnep. or ‘Siam tulip’, in family Zingiberaceae is a
native plant in Thailand. It is a high potential crop for cut flower and potted
plant. Thailand exports about two million rhizomes per year to Japan, EU
and USA.

Generally, flower and rhizome production starts from April to May,
the plant flowers in July to August during the rainy season in Thailand,
when the weather is averaging 27 to ee. 12 tol3 hrs of sunshine duration,
and 80% RH. Then, it becomes dormant in November to December, the
rhizomes are harvested in December to February when the temperature is
about 30/16 C (max/min), sunshine is about 10 hrs, and relative humidity
(RH) is about 65 to 70%. High demand of flower in the world market is
mostly in winter, when the environmental conditions, such as, short day
length and low temperature in winter are limiting factors for growth and
development of this plant.

All plants need light to use nutrients and manufacturing food.
Artificial light is useful when natural light is insufficient. Plant absorbs red
and blue lights, both are used in controlling photosynthesis, leaf development
and flowering. Incandescent light can supplement natural day light and give
a large amount of red light and infrared light (Barkley, 2005).

The responses of plants to day length were classified in three classes
i.e. short-day plants (SD), long day plants, and day length neutral plants;
however, this original classification has since become considerably more
complex with various subclasses. Plants differ in respect of the strictness of
dependence on day length were divided into, 1.e., (1) qualitative or obligate
photoperiodism, where there is an absolute requirement for a particular day
length (SD or LD plants), and (2) quantitative or facultative photoperiodism,
where a particular day length advances or enhances flowering, but the
plants will eventually flower anyway (Hart, 1988). Interruption by light of
dark period, called night break, can lead to floral promotion of LD plants
(Thomas and Vince-Prue, 1997). Hagiladi et al. (1997) reported that Cucurma
alismatifolia should be classified as quantitative long day plants, since long
day condition using supplement light source enhances flowering of this
plant. Therefore, the research was aimed to study the effect of planting date
and night break treatment on growth and development of C. alismatifolia
using incandescent light to extend flower production period from the rainy
season to winter.

Off-Season Flowering in Curcuma alismatifolia 175

Materials and Methods

Stubbed rhizomes with storage roots of Curcuma alismatifolia were stored
in cool room at 15 C, RH from 70 to 80% for 6 months from February to
July. The experiment using the storage rhizomes was started from August
to November, plants were grown in different conditions at four different
planting dates, 9 August; 9 September; 9 October; and 9 November. Before
planting, the rhizomes were soaked in water for 3 days to stimulate sprouting,
and planted in 6 x 12 inches plastic bags using sand : rice husk : rice husk
charcoal (1:1:1) as planting medium. Water was supplied daily and nutrient
solution containing, 200 mg! of N,50 mg | of P, 200 mg | of K, 65 mg
Is of Ca, 20 mg | of Mg,0.22 mg1_ioof B, 0.54mgl of Mn, 0.26 mg
bidtot Zn,0.04mgl of Moand0.45mgl_ of Fe was supplied twice a
week. For each planting date there were three treatments: T1, night break
treatment started from shoot emerged until the flower spike reached one
inch long; T2, night break treatment started from shoot emerged until the
first floret opened; and T3, control treatment where plants were grown in
natural conditions with no night break treatment. The growing plants were
exposed to 2 hrs supplement light from 20.00-22.00 hrs. Light source was
100 watt of incandescent light bulbs emitting about 462 umols m . Since
growth rate of plants were different at different planting dates, therefore T1
and T2 had different light supplement duration depending on the planting
dates as shown in Table 1. Plant growth in terms of plant height, number
of plants per cluster, and flower quality were collected. The experimental
design was a completely randomized design with 10 replications/treatment.

Table 1. Growing time required for starting night break treatments in Tl and T2 from
different planting dates.

Time required after planting (wks)

Planting dates T1 T2
(from planting to one inch of (from planting to opening
flower spike appeared) of the first floret)
Aug. 9 9 11
Sep. 9 10 12,
Oce> 12 14

Nov. 9 14 16

176 Gard. Bull. Singapore 59 (1&2) 2007

Results and Discussion

Plant growth

The results showed that growing habits of C. alismatifolia were not
significantly different at planting dates of 9 Aug. and 9 Oct. On the other
hand, they were affected by the night break treatments T1 and T2 compared
with control (T3) when planted in 9 Sep., and 9 Nov. Heights of plants at
late planting dates (9 Sep., 9 Oct. and 9 Nov.) were lower than early planting
date in 9 Aug. (Table 2). Assuming that the average temperature in Thailand
during that period was 20 to 24'C, which was cooler (26 to 27'C) than the
other periods and sunshine duration was from 10 to 11 hrs (Table 3). Lower
temperature during later planting dates has a deleterious effect on final plant
growth and development. However, night break treatment could stimulate
plant height compared with the control (Table 2).

Table 2. Plant height (cm) affected by night break treatments from different planting dates,
12 WAP.

Treatments Planting dates
Aug.9 Sep.9' Oct.9 Nov.9’

T1. night break until one inch of spike appeared 47.65 48.12a 42.70 34.40a

T2. night break until the first floret opened 50.08 55.62a 39.68 35.25a
T3. control (no night break) 45.08 32.48b 39.05 24.88b
LSD .05 ns 11.12 ns 6.38

‘ Means followed by different letters within the same column are significantly different
among treatments; ns: not significantly different.

Table 3. Meteorological data during August-December in Chiang Mai Province at the
Multiple Cropping Research Station, Chiang Mai University.

Month P Data
Air temp. ( C) _ Sunshine duration (hrs)
max Min avg
August 32 23 27 12
September 32 Zo es 12
October Sz 2A 26 | 11
November 31 19 24 11
December 29 13 20 10

Number of plants per cluster indicated the yield of rhizomes after
harvest. The results showed that night break did not affect the number of
plants per cluster of plants growing on 9 Aug., 9 Sep., and 9 Oct. (Table

Off-Season Flowering in Curcuma alismatifolia 77

4). However, the number of plants per cluster of the controlled treatment
T3 was significantly lower than T1 when planted on 9 Nov. (Table 4). This
indicates that the effect of night break was sensitive to low temperature
during the growing period.

Table 4. Number of plants per cluster affected by night break treatments from different
planting dates, 12 WAP.

Treatments Planting dates
Aug.9 Sep.9 Oct.9 = Nov.9!
T1. night break until one inch of spike appeared 125 L7s tS 2.75a
T2. night break until the first floret opened 1.50 1.00 £50 1.50ab
T3 control (no night break) 1.50 r25 S50 0.75b
LSD .05 ns ns ns 1.98

' Means followed by different letters within the same column are significantly different
among treatments; ns: not significantly different.

Days to flowering

The number of days from planting to flowering tended to be delayed when
plants were grown under night break treatments at the 9 Sep. and 9 Oct.
planting dates. However, days to flowering in the 9 Nov. group was less (99.25
days) for T1 compared to the T3 control treatment (103 days) (Table 5).
Night break treatments in T1 and T2 also increased flowering percentages,
compared to the control (Table 6). The similar results were also found in
Cosmos atrosanguineus (Hook.) Voss. (Kanellos and Pearson, 2000), Petunia
x hybrida (Adams et al., 1999) and Eustroma grandiflorum (Raf.) Shinn.
(Islam et al., 2005), the quantitative (facultative) long-day plants whose
flowering was advanced and hastened by long day.

Table 5. Number of days to the first floret opening affected by night break treatments from
different planting dates.

Treatments Planting dates
Aug.9 Sep.9 Oct.9 Nov. 9!
T1. night break until one inch of spike appeared 68.75 85.75 98.00ab 99.25b

T2. night break until the first floret opened GO715 89.50 102.50a 102.00ab
T3 control (no night break) 70.00 ADD 90.00b 103.00a
LSD .05 ns ns 12.44 2:0)

' Means followed by different letters within the same column are significantly different
among treatments; ns: not significantly different.

178 Gard. Bull. Singapore 59 (1&2) 2007

Table 6. Flowering percentages affected by night break treatments from different planting
dates.

Treatments Planting dates
Aug. 9 Sep.9 Oct.9 Nov. 9
T1. night break until one inch of spike appeared 80.00 80.00 80.00 60.00
T2. night break until the first floret opened 86.70 100.00 66.70 86.70
T3. control (no night break) 66.70 53.30 46.70 26.70

Flower qualities
Flower qualities in terms of spike length, number of coma bracts, number
of green bracts and length of flower stalk were determined. It showed that
both of the night break treatments (T1 and T2) did not affect all flower
quality parameters of the plants grown at 9 Aug. (Table 7 to 10). On the
other hand, they increased length of spike in general (Table 7), number of
coma bract (Table 8, Fig. 1) and length of flower stalk (Table 9) compared
to the control treatment at 9 Sep., 9 Oct. and 9 Nov. planting dates. Length
of spike in T1 were 14.05, 12.18 and 10.15 cm at 9 Sep., 9 Oct. and 9 Nov.,
respectively and they were not significantly different from T2, but they were
significantly higher than control treatment (T3) (Table 7). Number of coma
bracts were significantly higher in T1 (10.75, 12.00 and 10.50 bracts per spike
at 9 Sep., 9 Oct. and 9 Nov., respectively) and T2 (13.00 and 10.75 bracts at
9 Sep. and 9 Nov., respectively) than control treatment (T3) (Table 8). The
results of flower stalk length were similar to length of spike (Table 9). Chang
(2000) also reported that to extend flowering period of C. alismatifolia in
Taiwan using plastic tunnel and light illumination from 22.00 p.m. to 2.00 a.m.
increased quality on length of flower stalk, diameter of stalk and number of
coma bract. However, number of green bracts was not significantly different
among treatments at each planting date (Table 10). Later planting dates had
adverse effect, giving less flower qualities although the plants were supplied
with night break and also affected flower morphology as showed in Fig. 1.
For short day plant, such as chrysanthemum, night break is used for
floral bud initiation. In case of C. alismatifolia, a quantitative long-day plant,
night break seems to involve in extending photosynthetic period and stored
assimilates required for growth and flowering.

Off-Season Flowering in Curcuma alismatifolia 179

Table 7. Length of spike (cm) affected by night break treatments from different planting
dates.

Treatments Planting dates
Auig-9' ~ Sep" “Oct/9? "Now 9*
T1. night break until one inch of spike appeared 17.13 14.05ab 12.18ab 10.15a

T2. night break until the first floret opened 16.63 16.48a 12.45a 9.32a
T3. control (no night break) 17.38 13.45b 11.10b i220
LSD .05 ns 21a 1.26 0.97

' Means followed by different letters within the same column are significantly different
among treatments; ns: not significantly different.

Table 8. Number of coma bracts affected by night break treatments from different planting
dates.

Treatments Planting dates

Aug.9 Sep.9' Oct.9* = Nov.9’
T1. night break until one inch of spike appeared 12.50 10.75ab = 12.00a 10.50a

T2. night break until the first floret opened 12.00 13.00a 9:25D 10.75a
T3. control (no night break) iN 2825) 8.75c 9.50b 8.25b
LSD .05 ns 1.98 93 4.57

" Means followed by different letters within the same column are significantly different
among treatments; ns: not significantly different.

Table 9. Length of flower stalk (cm) affected by night break treatments from different
planting dates.

Treatments Planting dates
Aug.9 Sep.9* Oct.9' Nov. 9!
T1. night break until one inch of spike appeared 40.25 54.38ab = 31.25a 31.30a

T2. night break until the first floret opened 42.13 62.38a 28.20a 28.38a
T3. control (no night break) 40.50 43.38b = 22.75b 16.62b
LSD'.05 ns 12.60 529 6.13

' Means followed by different letters within the same column are significantly different
among treatments; ns: not significantly different.

180 Gard. Bull. Singapore 59 (1&2) 2007

Table 10. Number of green bract affected by night break treatments at different planting

dates.
Treatments Planting dates
Aug,9 Sep.9' Oct.9' ~~ Nov.9’
T1. night break until one inch of spike appeared = 8.75 9.00 8.75 7.00
T2. night break until the first floret opened 8.75 OS 8.25 2
T3. control (not supplied night break) 10.00 9.50 8.50 7.00
LSD .05 ns ns ns ns

' Means followed by different letters within the same column are significantly different

among treatments; ns: not significantly different.

a) planted on 9 Aug., flowered in late b) planted on 9 Sep., flowered in beginning

Oct. of Dec.

ce
c) planted on 9 Oct., flowered in d) planted on 9 Nov., flowered in beginning
beginning of Jan. of Feb.

Figure 1. Flower qualities influenced by night break and planting dates.

Off-Season Flowering in Curcuma alismatifolia 181

Conclusion

It was possible to produce off-season flower of Curcuma alismatifolia by
storing rhizome ina controlled room at 15 C, then stimulate shoot emergence
by soaking the rhizome in water for three days. It was not necessary to supply
night break when planted on August 9. However, for delayed growing in
September to November, night break treatment was necessary to promote
flowering, flowering percentage and increased flower qualities in December
and January. Duration of night break treatments between T1 and T2 were
not significantly different, therefore night break should be supplied from
shoot emerged until one inch of tight flower spike appeared (T1) which was
sufficient for off-season flowering.

Acknowledgements

The research was supported by the National Research Council of Thailand
(2004). We would like to thank H.M. the King’s Initiative Centre for Flower
and Fruit Propagation for cold room supporting. Thank to the Hitachi
Scholarship Foundation for financial support of the oral presentation in this
symposium.

References

Adams, S.R., S. Pearson, P. Hadley and W.M. Patefield. 1999. The effect of
Temperature and light integral on the phases of photoperiod sensitivity
in Petunia x hybrida. Annals of Botany 83: 263-269.

Barkley, S. 2005. House Plants: Aritficial Light at - http://www.agric.gov.
ab.ca/$development/septdocs.nsf/all/webdoc1380.

Chang, C. S. 2000. Dormancy in Curcuma (C. alismatifolia) at http://www.
chang.htm. 17/7/2004.

Hagiladi, A., N. Umiel and X.-H. Yang. 1997. Curcuma alismatifolia. I.
Effects of temperature and daylength on the development of flowers and
propagules. Acta Horticulturae 430: 755-761.

Hart, J.W. 1988. Light and Plant Growth. Urwin Hyman Ltd. London.
204 pp.

182 Gard. Bull. Singapore 59 (1&2) 2007

Islam, N., G.G. Patil and H.R. Gislerod. 2005. Effect of photoperiod and
light integral on flowering and growth of Eustoma grandiflorum (Raf.)
Shinn. Scientia Horticulturae 103: 441-451.

Kanellos, E.A.G and S. Pearson. 2000. Environmental regulation of
flowering and growth of Cosmos atrosanguineus (Hook.) Voss. Scientia
Horticulturae 83: 265-274.

Thomas, B. and D. Vince-Prue. 1997. Photoperiodism in Plants. Academic
Press. London. 428 pp.

Gardens’ Bulletin Singapore 59 (1&2): 183-188. 2007 183

Effects of 2,4-D on Callus Induction from Leaf Explants
of Cornukaempferia larsenii P. Saensouk

P. SAENSOUK, P. THEERAKULPISUT, B. KISWIJAN
AND S. BUNNAG

Applied Taxonomic Research Center, Department of Biology, Faculty of Science,
Khon Kaen University, Khon Kaen 40002, Thailand.
Email addresses: saensouk@yahoo.com, piythe@kku.ac.th,
bookij@kku.ac.th, sumbun@kku.ac.th

Abstract

Callus was induced from young leaves of Cornukaempferia larsenii P.
Saensouk on Murashige and Skoog medium supplemented with 3% sucrose
and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in
light and dark conditions. The highest number of callus formation, percentage
of callus formation and average weight of callus were obtained from young
leaves cultured on the medium supplemented with 0.5 mg/l 2,4-D in the
light condition. The callus could not be regenerated to plantlets in media
added with various concentrations of NAA and BA.

Introduction

The genus Cornukaempferia Mood & K. Larsen is the new genus in Zingib-
eraceae from Thailand, described by Mood and Larsen (1997, 1999). Two
species, C. aurantiflora Mood & K. Larsen and C. longipetiolata Mood &
K. Larsen, have been recognized. This genus is listed as rare and endemic
to Thailand and its distribution is restricted to only few provinces in the
northeastern and northern part of the country. C. aurantiflora has been used
by local people in northeastern Thailand to treat infected hemorrhoids and
laryngitis common in Thai children. During a floristic survey carried out in
May of 2005, a morphologically distinct species of Cornukaempferia was
discovered and will be named C. Jarsenii in honor of Professor Kai Larsen,
University of Aarhus, Denmark (Saensouk et al., 2007).

The new species is propagated vegetatively by pieces of rhizomes. In
a vegetatively propagated plant like Cornukaempferia, the risk of systemic
infections with rootknot nematodes, bacterial wilt and Fusarium from the
propagules is very high. Thus, the application of tissue culture can be used
to produce large amount of disease-free plantlets. The objective of this work

184 Gard. Bull. Singapore 59 (1&2) 2007

is to establish a system for vegetative propagation of this rare plant species
through tissue culture. This is the first report of callus induction from leaf
tissue of plants in this genus.

Materials and Methodology

Young leaves of C. larsenii (Fig. 1) collected from natural habitats were
washed with running tap water, rinsed with 70% (v/v) ethyl alcohol for 30
seconds, sterilized with 0.9 % sodium hypochlorite containing 2 drops of
Tween 20 for 15 seconds followed by three washes with sterilized distilled
water. The young leaves were cut into 1x1 cm pieces and cultured on MS
medium (Murashige and Skoog, 1962) supplemented with 3% sucrose,
0.7% agar, and 0, 0.1, 0.5, 1, 2, 3, 4 and 5 mg/l 2,4-dichlorophenoxyacetic
acid (2,4-D) in both light and dark conditions for 16 weeks. Callus was
transferred to regeneration medium, 1.e., MS medium, added with 0, 1, 3
and 5 mg/l 1- naphthaleneacetic acid (NAA) and benzyladenine (BA) for
16 weeks. The cultures were incubated at 25 + 2°'C under white, fluorescent
light (2,000 lux) at a 16 h photoperiod or in the dark. All the experiments
were conducted using complete randomized design (CRD) with 15 replicates
each containing one explant per culture tube. Data were analyzed using
ANOVA and the mean separation was achieved by the Duncan’s Multiple
Range Test (DMRT). The test of statistical significance was performed at
the 5% level using the SPSS program (version 11.5).

yer > \ ) #

x
oe

Figure 1. Cornukaempferia larsenii. A. Habit; B. Flower. (Scale bars A= 10 cm and B= 1 cm).
Results and Discussion

Preliminary efforts to propagate this plant by culturing shoot tips from
underground stems resulted in extremely high level of contamination,
therefore, an attempt was made using the leaf blade as explants. Young leaves
from natural habitats were cultured on MS medium with various levels of

Effects of 2,4-D on Callus Induction of Cornukaempferia larsenii 185

2,4-D for induction of callus in the light and dark conditions. The callus was
soft in texture, friable in structure, and yellowish white (Figs. 2 and 3). Callus
did not form on medium lacking 2,4-D. The callus formation occurred on
medium added with 0.1,0.5,1,2,3,4 and 5 mg/l 2,4-D in the light, and 0.5, 1,2
and 3 mg/| 2,4-D in the dark, after 8 weeks of culture. The highest percentage
of callus formation (99.33%) and the highest average weight of callus (2.61
g) were obtained from young leaves cultured on the medium supplemented
with 0.5 mg/12,4-D in the light condition for 16 weeks (Table 1). These results
differ from that of Babu et al. (1992) who reported callus formation on the
young leaves of Zingiber officinale Rosc. (ginger) cv. Maran cultured on
MS medium containing 2-5 mg/l 2,4-D. Kackar er al. (1993) induced callus
formation from young leaf segments of ginger on MS medium added with
dicamba. Samsudeen et al. (2000) induced ginger anther to develop callus on
MS medium supplemented with 2-3 mg/]2,4-D. Prakash et al. (2004) obtained
semi-friable callus from leaf sheath explants of Curcuma amada Roxb. on
MS medium added with 2 mg/1 2,4-D. Moreover, Salvi et al. (2001) induced
callus from leaf base of turmeric on MS medium supplemented with 2 mg/l
dicamba, 2 mg/] picloram or 5 mg/] NAA in combination with 0.5 mg/l BA.
Callus was induced more effectively in the light than in the dark condition.
These results differ from Malamug etal. (1991) who reported callus induction
from shoot tips of ginger on MS medium containing 1 mg/l BA and 0.5 mg/l
2,4-D in the dark condition. High contamination of cultures was reported
when rhizomes or vegetative buds are used as explants for initiation of
culture. By using the leaf tissue as explants this problem was eliminated
almost completely. In Cornukaempferia, 2,4-D was used for induction of
callus from leaf explants (see Table 1), but when callus was transferred to MS
medium added with varying concentrations of NAA and BA and cultured
for 16 weeks, plant regeneration failed. Varying types and concentrations
of auxin and cytokinin have been successfully used to regenerate plantlets
from calli of several other species of Zingiberaceae. In ginger, Malamug et
al. (1991) reported plant regeneration from shoot tip callus on MS medium
added with 1 and 3 mg/l 2,4-D. Callus could also be regenerated from the
young leaf explants of ginger on MS medium supplemented with 0.2 mg/l
2,4-D and 5 mg/I kinetin or 5 mg/] BA (Babu et al., 1992). Samsudeen et al.
(2000) was able to regenerate plantlets from callus of ginger anther on MS
medium supplemented with 5-10 mg/l BA and 0.2 mg/l 2,4-D. Prakash et al.
(2004) cultured semi-friable callus from leaf sheath explants of Curcuma
amada Roxb. on MS medium containing 2 mg/l BA and 0.5 mg/l NAA and
produced optimum shoot initiation and development. In addition, Salvi
et al. (2001) transferred callus of turmeric (Curcuma longa Linn.) to half
strength MS medium supplemented with 5 mg/l BA in combination with
TIBA or 0.1 mg/l 2,4-D, green shoot primordial were seen to differentiate

186 Gard. Bull. Singapore 59 (1&2) 2007

Figure 2. Callus induction from leaf explants of Cornukaempferia larsenii on MS medium
added with 0, 0.1, 0.5, 1,2, 3,4 and 5 mg/1 2,4-D in the light condition (scale bars = 2.5 cm).

Figure 3. Callus induction from leaf explants of Cornukaempferia larsenii on MS medium
added with 0.5, 1,2 and 3 mg/l 2,4-D in the dark condition (scale bars = 2.5 cm).

Table 1. Effect of 2,4-D on callus induction from leaf explants of Cornukaempferia larsenii
in the light and dark conditions for 16 weeks.

2,4-D No. of Light | Dark
(mg/l) explants
| No. of % of Average No.of | %of Average
callus callus weight of callus callus weight of
formation formation | callus (g) formation 'formation callus (g)
| | mean+SE -| | mean+SE
La ee 0 0 a* L a 0 0 a*
Qi? ergs). Steamy 20 2.13+0.25ab | Oa | 0 Oa
0.5 | a5 14d 93.33 | 2.61+0.40 d 4b | 26.67 | 2.20+0.29b
graf 146 fica 13.53°° "P23 020 40 sap’ =) 20 1.77+0.25 ab
2 ry? ts" be | 53.33 | 2.10+0.36 be (Sap 20 '1.40+0.40 ab
3 if, Tbe | 46.67 |2164035be | 2ab 5.33 | 1.00+0.14 ab
4 i Tbe | 46.67 |2.0640.38bc | 0a | 0 | Oa
5 NS Pa pe Pas” See Oe ees Oa

*In each column the values with the different letters differ significantly (P = 0.05) as
determined by DMRT (see text).

Effects of 2,4-D on Callus Induction of Cornukaempferia larsenii 187

from the surface of the callus. On transfer of regenerating cultures to half
MS media supplemented with kinetin, shoot primordial developed into well-
differentiated shoots. When shoots were transferred to medium devoid of
phyto-hormone, complete rooted plants were obtained. Further experiments
are being performed to obtain efficient plant regeneration using different
growth regulators and culture conditions.

Conclusion

This is the first report describing tissue culture of Cornukaempferia
larsenii, a recently discovered and rare species of Thailand. We reported
a successful protocol for the efficient and reliable callus induction using
cultured leaf explants of this species. Plant regeneration from leaf tissue
through an intermediary callus phase may be a possibility of increasing rate
of somaclonal variations that can be exploited for crop improvement which
are not available by conventional methods. Futhermore, regeneration of
plantlets from callus is an important technique, which can be utilized in the
application of tissue culture in developing new germplasm.

Acknowledgements

This study was supported by University Staff Development Program,
Mahasarakham University and Applied Taxonomic Research Center,
Department of Biology, Faculty of Science, Khon Kaen University,
Thailand.

References

Babu, K.N., K. Samsudeenn and M.J. Ratnambal. 1992. Jn vitro plant
regeneration from leaf-derived callus in ginger. Plant Cell, Tissue and
Organ Culture 29: 71-74.

Kackar,A.,S.R. Bhat, K.P.S.Chandel and S.K. Malik. 1993. Plant regeneration
via somatic embryogenesis in ginger. Plant Cell, Tissue and Organ Culture
32: 289-2972.

Malamug, J.J.F., H. Inden and T. Asahira. 1991. Plant regeneration and
propagation from ginger callus. Scientia Horticulturae 48: 89-97.

Mood,J.and K. Larsen. 1997. Cornukaempferia,anew genus of Zingiberaceae
from Thailand. Natural History Bulletin of the Siam Society 45: 217-221.

188 Gard. Bull. Singapore 59 (1&2) 2007

Mood, J.and K. Larsen. 1999. New to cultivation: the genus Cornukaempferia
in Thailand with description of a second species. The New Plantsman 6:
196-205.

Saensouk,P.,P.Theerakulpisut and P. Chantaranothai,2007.Cornukaempferia
larsenii sp. nov. (Zingiberaceae): A new species from Thailand. The
Natural History Journal of Chulalongkorn University 7: 169-173.

Salvi, N.D., L. George and S. Eapen. 2001. Plant regeneration from leaf base
callus of turmeric and random amplified polymorphic DNA analysis of
regenerated plants. Plant Cell, Tissue and Organ Culture 66: 113-119.

Samsudeen, K., K.N. Babu, M. Divakaran and P.N. Ravindran. 2000. Plant
regeneration from anther derived callus cultures of ginger (Zingiber
officinale Rosc.). Journal of Horticultural Science & Technology 75: 447-
450.

Gardens’ Bulletin Singapore 59 (1&2): 189-202. 2007 189

Evaluation of anti-oxidant and cytotoxic properties of
tropical ginger, Zingiber montanum (J. Konig) A. Dietr.

G.J. SHARMA ', P. CHIRANGINI 'AND K.P. MISHRA ”

, Department of Life Sciences, Manipur University, Imphal—795003, India
* Radiation Biology & Health Sciences Division, Bhabha Atomic Research
Centre, Mumbai—400085, India

Abstract

Many members belonging to the family Zingiberaceae are well known for
their uses in traditional medicine for curing various ailments since times
immemorial. The rhizomes of some medicinal Zingiberaceae are widely used
in the dietary intakes. Curcumin present in turmeric and gingerol in ginger
have been known to possess anti-oxidant properties. The northeast India,
which lies within the Indo-Burmese mega-biodiversity ‘hotspot’ region, is
a genetic treasure house of biological resources with good representation
of Zingiberaceous species. The present studies were conducted to assess
the free radical scavenging antioxidant properties of rhizome extract of
Zingiber montanum (J. Konig) A. Dietr [=Z. cassumunar Roxb.] using
various chemical assay systems like diphenyl picrylhydrazyl (DPPH),
superoxide (O, ) and hydroxyl (OH) radical scavenging methods. Increased
percent of DPPH decoloration from 50-500 ug/ml indicated concentration
dependent scavenging activity of DPPH radicals by the crude extract of
this species. Even at a low concentration of 1 ug/ml, the rhizome extract
showed strong (~75%) OH scavenging activity. Similarly, the crude extract
showed a concentration dependent inhibition of O, radical production
where a concentration of 50 ug/ml almost showed 100% inhibition.
Cytotoxicity was assessed by MTT assay using NIH 3T3 fibroblast cell line.
Only 28% cytotoxicity was observed up to a concentration of 100 ug/ml.
The results strongly support the therapeutic use of crude rhizome extract
of Z. montanum for its dietary intake and use as traditional medicine,
thereby suggesting its potential as promising radioprotective agent.

Introduction

Manipur, which lies within the Indo-Burmese mega-biodiversity ‘hotspot’
region in the northeast India, is a genetic treasure house of rich biological
resources. This active ‘centre of speciation’ represents a zone of gene diversity

190 Gard. Bull. Singapore 59 (1&2) 2007

for a variety of wild as well as domesticated plants, and a secondary centre
for several economically important medicinal and aromatic plants. These
gene pools are invaluable resources and their sustainable utilization can,
through biotechnological interventions, bring about economic growth of the
region. Bioprospects of these biological wealth scattered in the potentially
useful plants as ‘bio-active molecules’ need to be fully explored. These are
the molecules, which would help in designing and manufacturing various
plant-based drug formulations.

A wide variety of phytochemicals including polyphenolics,
carotenoids, terpenoids, coumarins, saponins, phytosterols, curcuminoids,etc.,
have been identified in several plants. The most publicized phytochemicals
with antioxidant profiles have been vitamins C, E and beta-carotene.
Flavonoids are widely distributed in plants and other plant products, and
are powerful inhibitors of lipid peroxidation, reactive oxygen species (ROS)
scavengers, inhibitors of damage by haem protein/ peroxide mixtures, metal
ion binding agents and inhibitors of lipoxygenase and cyclooxygenase
enzymes in vitro. The degree of hydroxylation and relative position of -OH
groups are of prime importance in determining antioxidant activity. In whole
animals, flavonoids have been reported to exert anti-inflammatory and anti-
cancer effects (Read, 1995).

Frankel et al. (1995) reported that plant phenols in red wine exerted
cardioprotective effect. Keli et al. (1996) suggested an inverse relationship in
the incidence of coronary heart disease and stroke in elderly men with dietary
intakes of flavonoids from tea, fruits and vegetables in human populations.
Phenolic substances have been found to possess anti-carcinogenic and anti-
mutagenic activities, the majority of these naturally occurring phenolics
retain antioxidative and anti-inflammatory properties which appear to
contribute to their chemopreventive or chemo-protective activity (Surh,
1999). The human body is constantly under attack from free radicals, which
are highly reactive chemical entities and are fundamental to any biochemical
processes representing aerobic life. They are continuously produced by the
body’s normal use of oxygen, such as respiration and cell-mediated immune
functions, and are generated through a variety of environmental agents.
Free radicals can react readily with various biomolecules, such as DNA,
proteins and lipids, to cause cellular lesions, which, in turn, lead to various
human diseases (Halliwell and Gutteridge, 1999). In vitro generated sulfur
free radicals have been suitably used in experiments against a reference
molecule such as curcumin, beta-carotene or retinol (Devi et al., 1992;
D’ Aquino et al., 1994) for rapid evaluation of antioxidant potentials.

The rhizomes of tropical ginger, Zingiber montanum (J. Konig) A.
Dietr. (syn. Z. cassumunar Roxb.), abundantly found in Manipur are used
in diarrhoea, colic, and used as stimulant, carminative, for flavouring food

Anti-oxidant and cytotoxic properties of tropical ginger 191

preparations and substituting for true ginger as antidote for snakebites,
and in asthma, ascites, anemia, bruises, bronchitis, dropsy and fever, and
for treatment of intestinal disorders, swellings, rheumatism, numb feet and
painful parts. Antioxidant molecules already reported from this plant are
alflabene, cassumunene, cassumunaquinones I, II, cassumunins A, B, C and
cassumunarins A, B, C (Dinter et al., 1980; Masuda and Jitoe, 1994; Jitoe et
al., 1992). Our investigations on sulfur free radical reactivity using curcumin
as a reference indicator, and its inhibition by various crude extracts
of fourteen medicinal Zingiberaceous species in vitro showed that Z.
montanum [as Z. cassumunar in the work] exhibited maximum antioxidant
property (Chirangini et al., 2004). In this paper, attempts have been made to
screen antioxidant potentials using DPPH, hydroxyl and superoxide radical
scavenging assays and cytotoxicity using NIH 3T3 mouse fibroblast cell
lines.

Materials and Methods

The tropical ginger, Zingiber montanum, has rootstocks that are perennial.
Rhizomes are bright yellow inside with strong camphoraceous scent. Leaves
are oblong-lanceolate and hairy underneath. The spike-like inflorescence
is oblong with ovate, reddish bracts. From the bracts, pale yellow colored
flowers come out in acropetal succession. The corolla tube is as long as
the bract with whitish segments, upper portion being broader and more
concave. The most beautiful part of the flower, the lip or the labellum, is
yellowish white in coloration with a deeply bifid midlobe. The basal auricles
are large, oblong, and obtuse. The male part of the flower, the stamens are
yellowish white, but shorter than the lip. The ovary is 3-celled and the ovules
are many arranged in the inner angle of the cells. Plants collected from
wild natural wetland habitats of Manipur grown in the Experimental Field,
Department of Life Sciences, Manipur University, since July 2000, were
used in these experiments. Morpho-taxonomic characters were properly
recorded. Healthy, uninfected and unbruised fresh rhizomes were used for
all the experiments. Herbarium voucher have been collected and deposited
at Herbarium of Manipal University, Imphal (MU/LSD/Herb.32): India,
Manipur, Imphal, Thoubal & Bishenpur Districts, 22.V1I.2000, Chirangini et
al. 32.

Preparation of the Zingiber montanum extract

Fresh rhizomes were washed and cleaned thoroughly in running tap water.
The roots were removed along with the outer scales. After drying in between
the folds of the filter paper, rhizomes were weighed and crushed with the

192 Gard. Bull. Singapore 59 (1&2) 2007

help of mortar and pestle. Then, it was homogenized in absolute methanol
(1gm/ml). The crude extracts obtained were centrifuged twice and filtered,
using Whatman No. | filter paper, till a clear supernatant was obtained. The
supernatant was vacuum evaporated till dryness. The residue obtained was
kept at 4°C for future use.

I. Antioxidative capacity - The antioxidative capacity of Z. montanum ex-
tract was examined by comparing it to the activity of known antioxidants,
such as ascorbic acid, by the following chemical assays - scavenging of
DPPH radical and oxygen radicals such as superoxide, and hydroxyl radicals.

DPPH assay

The DPPH assay was carried out as described by Cuendet et al. (1997) with
slight modification. The reaction mixture consisted of 250 uM DPPH in 100%
methanol with 50-500 ug/mL of the crude extract of Z.montanum or 0.01-0.1
mM of vitamin C. After a 30-min incubation period in the dark at room
temperature, the absorbance was read against a blank at 517 nm. Percentage
inhibition was determined by comparison with a methanol treated control
group. The percentage of DPPH decoloration was calculated as follows:

% DPPH decoloration = [1 — O.D.sample/ O.D. control] x 100

The degree of decoloration indicates the free radical scavenging efficiency
of the substances. Values are presented as mean + standard deviation of
three determinations.

Hydroxyl radical scavenging activity assay

Hydroxyl radical scavenging activity assay was carried out by measuring
the competition between deoxyribose and the extracts for hydroxyl radicals
generated from the Fe(II)/ascorbate/EDTA/H,O, system. The attack of
the hydroxyl radical to deoxyribose leads to thiobarbituric acid reactive
substances (TBARS) formation (Kunchandy and Rao, 1990). Various
concentrations of the extracts were added to the reaction mixture containing
2.8 mM deoxyribose, 25 uM FeCl, 100 uM EDTA, 100 uM ascorbic acid,
2.8 mM H,O., and 5 mM phosphate buffer (pH 7.4), making up a final
volume of 1.0 mL. The reaction mixture was incubated at 37 °C for 1 h. The
formed TBARS were measured by the method of Ohkawa et al. (1979).
One milliliter of thiobarbituric acid (TBA, 1% w/v in 50 mM NaOH) and
1 mL of trichloroacetic acid (TCA, 2.8% w/v) were added to test tubes and
incubated at 100°C for 30 min. After the mixtures cooled, absorbance was
measured at 532 nm against a blank containing deoxyribose and buffer.
Reactions were carried out in triplicate.

Anti-oxidant and cytotoxic properties of tropical ginger 193

Inhibition (/) of deoxyribose degradation in percent was calculated
in the following way:

= (Ao- A,)/Ao x 100

where Ay is the absorbance of the control reaction (containing all reagents
except the test compound) and A; is the absorbance of the test compound.

Inhibition of superoxide radical

Superoxide radical generated by the hypoxanthine/xanthine oxidase system
was determined spectrophotometrically by monitoring the product of
nitroblue tetrazolium (NBT). Various concentrations of the extracts were
added to the reaction mixture containing 100ml of 30 mM EDTA (pH 7.4),
10ml of 30 mM hypoxanthine in 50 mM NaOH, 200ml! of 1.42 mM NBT
and the final volume of 3 ml was made up by 50 mM PO4 Buffer (pH 7.4).
After adding 100ml of 0.5 U/ml xanthine oxidase, the reaction mixture
was incubated for 30 min at 25°C. The absorbance was read at 560 nm and
compared with control samples in which the enzyme, xanthine oxidase, was
not included.

The percent inhibition of superoxide radicals was calculated from the optical
density of the treated and control samples.

Inhibitory effect (% ) $ [(Aseo control A560 sample) A.s6o eanttell x 100

II. Cytotoxicity studies - /n vitro cytotoxic effect of crude extracts of Z.
montanum was studied on normal mouse embryo fibroblast cell (NIH/3T3).
The methanol extract was dissolved in dimethylsulphoxide (DMSO). The
cell line NIH/3T3 was provided by National Centre for Cell Science, Pune,
India.

Cell culture conditions

Stock cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
with 10% fetal calf serum supplemented with 0.04M NaHCOs, 0.006%
penicillin and 0.025% streptomycin at 37 C in an atmosphere of 5% CO,
and 95% humidity. The medium was changed every three days. Monolayer
cells were plated out at 2x10* cells/well in 96-well microtitre plate. The cell
growth was found to be exponential during 2-3 days in the medium.

MTT assay

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] easily

194 Gard. Bull. Singapore 59 (1 &2) 2007

enters cells. The esterases present in viable cells can cleave MTT to form
purple-colored formazan crystals which are then solubilized. Color produced
is directly proportional to cell viability.

The cytotoxic effect of the crude extracts, expressed as cell viability, was
assessed by MTT staining experiment (Mosmann, 1983). Briefly, confluent
cultures of NIH/3T3 cells were treated with medium containing the
methanol extract of Z. montanum at concentrations from 2-400 ug/mL. The
extract was first dissolved in absolute DMSO and then in DMEM. The final
concentration of DMSO in the test medium and controls was <1%. Cells
were exposed for 48 hr to test medium with or without the extracts. The
medium was removed and 100 ul of MTT solution (1 mg/ml in PBS) was
added to each well of 96 multiwell plates and the plates were incubated for
additional 3 hr at 37 C. Finally, 100 pl of 10 % (w/v) sodium dodecyl sulfate
in 0.01N HCl was added to each well and the absorbance was measured
at 550 nm using the ELISA reader (Biotek System). Each concentration
of plant extract was tested in hexaplicate and repeated twice in separate
experiments.

Percentage viability was calculated from the following relation:

% Viability = [1 — OD sampie/OD contrat] X 100

Results and Discussion

DPPH Assay: As shown in Fig. 1, Vitamin C and methanol extract of Z.
montanum were able to reduce the stable radical DPPH to the yellow-col-
ored diphenylpicrylhydrazine. The strongest effect was observed in 0.1mM
Vitamin C with 91% DPPH decoloration. Up to 86% DPPH decoloration
was observed in case of 500 ug/mL Z. montanum extract that is having
similar effect with 0.08mM Vitamin C (84%).

Hydroxyl radical (OH) scavenging: When the methanol extract of Z.
montanum was incubated with the reaction mixture used in the deoxyribose
degradation assay, they removed hydroxyl radicals from the sugar and
prevented its degradation. As shown in Fig. 2, Z. montanum extract, even
at a concentration of 1 ug/mL there is 59% scavenging of the OH radical
showing very potent activity. The following results show that there is no
dose dependent reponse for OH radical scavenging capacity.

Anti-oxidant and cytotoxic properties of tropical ginger

100

DPPH decoloration (%)

ff fa gl UJ
i af sf i
go" A oe" ot

Concentration of Vit C

DPPH decoloration (%)

195

(b)

| itl | yal al
ml A hu L N
i" 9 (ud 9 rT
sa UF oY ou vl qi!

30 (0 30 ao 30

Concentration of Z. cassumunar rhizome extract

Figure 1. DPPH radical scavenging activity of (a) Vitamin C and (b) Z. montanum rhizome.

OH radical scavanging activity (%)

ee ae ae
qi qi lea
spt ag! Pe ow

l
yi! qi"
0 ud aoe EP
2"

| m |

Figure 2. Hydroxyl radical (OH ) scavenging activity of Z. montanum rhizome extract.

Inhibition of superoxide radical: Methanol extract of Z. montanum were
found to scavenge the superoxide radicals generated from the hypoxanthine/
xanthine oxidase method. There is a dose dependent response of the
compound as well as the extract (fig. 3). The results do point towards an
increased trend in the response with small increases in the concentration of

the extract.

Cytotoxicity testing by MTT assay: Methanol extracts of Z. montanum tested
for cytotoxicity against normal mouse fibroblast cell line using standard
MTT assay showed very low toxicity up to 100ug/ml (fig.4). Only 24% of
the cells survive the toxic effect of Z. montanum extract at a concentration

of 200 ug/mL.

196 Gard. Bull. Singapore 59 (1&2) 2007

100

Inhibitory activity (%)
ch ~]
oO th

Ni
nh

| | al | |
ar? am im tb il in
co aud 3? uy que gn?
Concentration of 2. cassuymunar extract

Figure 3. Inhibition of superoxide radical production by Z. montanum rhizome.

100

|
th

Viability of NIH 3T3 cells (%)
th 3

pl al | nl |
‘ ! a f 4 uv
ious sou? cov" ;oou? aug

ont 59
id %

Concentration of 2. cassumunear rhizome extract

Figure 4. Cytotoxicity of methanolic extracts of Z. montanum rhizome.

Sulfur free radicals (GS) formed in gamma irradiated aqueous glutathione
(GSH) solution could easily oxidize the chrome orange-yellow compound
curcumin - its depletion increasing with increasing dose of radiation.
Supplementation of the crude rhizome extract reduced the depletion of
curcumin significantly. The inhibition of curcumin depletion in the rhizome
extract-added reaction solution varied with the species showing different
protective indices (PIs). A relative comparison of PIs at 75 Gy exposure
showed Zingiber cassumunar [currently accepted name Z. montanum|
> Kaempferia galanga > Hedychium flavascens > Zingiber officinale >

Anti-oxidant and cytotoxic properties of tropical ginger 197

Hedychium coccineum > Curcuma caesia > Curcuma amada > Alpinia
allughas > Curcuma leucorhiza > Hedychium coronarium > Alpinia galanga
(Chirangini et al., 2004).

Free radicals generated either by endogenous metabolism or external
environmental agents are harmful to cellular constituents, such as proteins,
lipids, DNA and carbohydrates, and result in possible alteration of cell
function (Davies et al., 1987; Dezwart et al., 1999; Gebicki & Gebicki, 1999).
ROS are known to be carcinogens and act at several stages in malignant
transformation (Cerutti, 1994), including permanent DNA _ sequence
changes in the form of point mutations, deletions, gene amplifications, and
rearrangements which may result in the activation of proto-oncogenes or
the inactivation of tumor-suppressor genes (Hsie et al., 1986; Moraes et al.,
1990). The role of these ROS in oxidative damage to the membranes and
mechanism of apoptotic death of thymocytes have been well elaborated
(Bhosle et al., 2002; Mishra & Hota, 2003; Pandey & Mishra, 2003).

The body’s antioxidant defense system is composed of various
antioxidants present in the plasma or biological fluids in a reduced form.
While scavenging/neutralizing the free radicals, they are either oxidized
or exhausted. An external anti-oxidant can prevent oxidative damage by
inhibiting the generation of reactive species, scavenging free radicals, or
raising the endogenous level of antioxidant defense. To maintain antioxidant
level in the body, external supplementation is necessary for healthy living
(Halliwell & Gutteridge, 1989). Supplementation of natural antioxidants
through a balanced diet could be more effective, and also more economical
than supplementation of an individual antioxidant, such as Vitamin C or E,
in protecting the body against oxidative damage under various conditions
(Wang et al., 1996). It has been known that several medicinal plants contain
‘active principles’ possessing antioxidant properties. In Manipur, a number
non-conventional and under-used plant-based food, particularly belonging
to the family Zingiberaceae, possessing rich antioxidant properties are
consumed by the people which perhaps may be the basis for low incidence
of cancers (Chirangini et al., 2004).

Although some work has been done on the radioprotective effect
of curcumin extracted from Curcuma longa (Inano & Onado, 2002) and
ginger rhizome (Jagetia et al., 2003), detailed studies have not been carried
out as yet on the potential antioxidant properties of Z. montanum. The
present studies made using the DPPH, hydroxyl and superoxide radical
scavenging assays, therefore, have reaffirmed the antioxidant potentials
of Z. montanum in a much more elaborate manner, and are hence quite
relevant. Although the crude extracts of these various plants have numerous
medicinal potentials, clinical applications can be made only after extensive
research on the bioactivity, mechanism of action, pharmaco-therapeutics

198 Gard. Bull. Singapore 59 (1&2) 2007

and toxicity studies of the different compounds present in these plants.
Recent years have seen an increased enthusiasm in treating various diseases
with natural products. Many phytonutrients or phytochemicals having very
high antioxidant profile need to be investigated for their application as anti-
tumour or radioprotective agents to inhibit acute and chronic effects and
even mortality after irradiation. It is expected that some novel compounds
may turn out to have very rich radioprotective property which could be
comparatively better than that of amifostine, the only agent that reduces
radiation induced toxicity during clinical trials.

It can be concluded that Z. montanum rhizome extract possesses
significant radical scavenging and anti-oxidant properties. Besides being
an efficient scavenger, cytotoxicity of Z. montanum rhizome extract above
100 ug/ml indicate its significant potential as an anti-tumor agent. The
results shown above do strongly support the therapeutic applicability of Z.
montanum extract for its dietary intake and use in traditional system of
medicine. Based upon these significant anti-oxidant properties, there is an
urgent need for investigation of the rhizome extract of Z. montanum for its
radioprotective activity using suitable in vivo mammalian test systems.

Acknowledgement

The authors are thankful to the Board of researches in Nuclear Sciences,
Department of Atomic Energy, Government of India, Mumbai for providing
financial support [vide Grant No. 2004/37/29/BRNS/2130]. We thank Dr.
Jana Leong-Skornickova (SING) for comments regarding the nomenclature
of the names Zingiber montanum and Z. cassumunar.

References

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Gardens’ Bulletin Singapore 59 (1&2): 203-220. 2007 203

The Genus Curcuma L. ( Zingiberaceae ):
Distribution and Classification with
Reference to Species Diversity in Thailand

P. SIRIRUGSA ', K. LARSEN * AND C. MAKNOI°

‘Faculty of Science, Prince of Songkla University, Hat Yai 90112, Thailand.
* Department of Systematic Botany, Aarhus University, Building 540,
Ny Munkegade 116, DK-8000 Aarhus C., Denmark.

* Queen Sirikit Botanic Garden, The Botanical Garden Organization,
P.O. Box 7, Maerim, Chiang Mai 50180, Thailand

Abstract

The genus Curcuma L. is one of the largest genera in the Zingiberaceae,
with about 80 species, and distributed throughout tropical Asia from India
to South China, Southeast Asia, Papua New Guinea and Northern Australia.
In Thailand, thirty-eight species have been found. Taxonomic knowledge of
this genus is necessary for citing correctly the species used commercially as
spices, ornamentals and medicines. Formerly, Curcuma was a member of the
tribe Hedychieae. According to the new classification of the Zingiberaceae
proposed by Kress et al. (2000), this genus belongs to the tribe Zingibereae.
This paper presents an overview of the genus Curcuma and its species
diversity in Thailand. The infrageneric classification of the genus based on
morphology and molecular evidences with reference to species diversity in
Thailand is discussed. The representative taxa, their distribution and uses
are provided.

Introduction

Curcuma is one of the largest genera in the Zingiberaceae which comprises
approximately 80 species (Larsen, 2005). It is widely distributed in the tropics
of Asia from India to South China, Southeast Asia, Papua New Guinea and
Northern Australia (Fig. 1). In Thailand they are normally found in the teak,
pine or dipterocarp forests at the altitude 500-900 m. Some species, such as
C. alismatifolia Gagnep., grows well in the open areas. The most common
species, C. parviflora Wall., grows in wide range of altitudes from 100 — 1300
m on limestone hills. Generally, most Curcuma grows well in loose and sandy
soil in shaded areas.

204 Gard. Bull. Singapore 59 (1&2) 2007

Pacific

(Ocea rt

Indian ¢

Figure 1. Distribution of the genus Curcuma

Morphological Characters of Curcuma

The habit of Curcuma is a rhizomatous herbaceous plant, comprising of
underground parts, leafy shoot and leaf blades (Fig. 2).

Underground parts. At the base of the aerial shoot, the stem consists of
erect ovoid or globose structure (primary rhizome), bearing few to many
horizontally branches, and roots. However, branched rhizomes are rarely
produced in some species. The roots often produce ellipsoid tubers. Inner
part of rhizomes varies in various colours, i.e., white, cream, yellow, orange,
blue and bluish-green. Some species have a unique colour of rhizomes
which are useful for identification, such as the bluish-green rhizome in C.
aeruginosa Roxb.

Leafy shoots. These are 1-2 m high, forming a pseudostem by the leaf-
sheaths and surrounded by the leafless sheaths at the base. Leaf blades are
usually large, lanceolate or elliptic, rarely linear with or without the purple
stripe along either side of the midrib.

Inflorescence. This occurs either terminally on the leaf-shoot, with the
peduncle enclosed by the leaf sheaths, or on the separate shoot with the
peduncle enclosed by the bladeless sheaths. The inflorescence can be
cylindric, conic or ovoid in shape.

Distribution and Species Diversity of Curcuma in Thailand 205

Figure 2. Curcuma. A. Habit; B. Primary rhizome with branches; C. Rhizome of C. caesia.

Bracts. Bracts are usually large and joined to each other forming pouches
at the base, the free ends of the bracts are normally wide spread, each
subtending a cincinnus of 2-10 flowers. In many species the uppermost bracts,
which are called “coma”, are longer than the rest and differently coloured.
They are usually sterile.

Flowers. Flowers are enclosed by bracteoles, comprising of the following
floral parts: Calyx is tubular, unequally toothed, deeply divided along one
side. Corolla-tube is more or less funnel shaped; corolla-lobes are unequal,
the dorsal slightly larger than the lateral ones, and its apex is hooded.
Staminodes are petaloid, elliptic, oblong or linear. Labellum has a thickened
middle part and thinner lateral lobes which overlap the staminodes. Stamen
has a short and broad filament, and a constricted apex. Anther is versatile,
with or without spurs, and the anther-crest is usually small. Spurs vary in
several shapes and sizes, and they are important characters for infra-generic
classification. Ovary is glabrous or pubescent, and 3-lobed. Stylodes can be
present. Capsule is ellipsoid, and seeds are arillate.

Flower forms (Fig. 3). The different arrangement of staminodes and corolla-
lobes made up the 2-formed flowers, 1.e., closed form: the staminodes are
wrapped by the dorsal corolla-lobe; and the open form: the staminodes are
free from the dorsal corolla-lobe.

206 Gard. Bull. Singapore 59 (1&2) 2007

Figure 3. Curcuma flowers, showing the closed form (left) and open form (right).

Distinguished Characters

It is easy to distinguish Curcuma from other genera of Zingiberaceae by the
following characters: the joining bracts to form pouches; flowers borne in
cincinni, subtended by bracteoles and bracts; and the sterile and differently
coloured coma bracts.

Classification of Zingiberaceae

Zingiberaceae (Burtt & Smith, 1972) Zingiberaceae (Kress et al., 2002)
Tribe Genus Subfamily Tribe Genus
Hedychieae Curcuma Siphonochiloideae Siphonochileae
Zingibereae Tamijioideae Tamijieae
Alpinieae Alpinioideae Alpinieae
Globbeae Riedelieae
Zingiberoideae Zingibereae Curcuma
Globbeae

According to the system of Burtt & Smith in 1972, which was
accepted for many years, the family Zingiberaceae was classified into 4
tribes and Curcuma was placed in the tribe Hedychieae. In the year 2002 a
new system was proposed by Kress et al. Zingiberaceae was classified into
4 subfamilies and 6 tribes (as shown above), and Curcuma has been placed
in the tribe Zingibereae.

The genus Curcuma shows great morphological variations, the
overlapping similarities among them made confusion in the identification
of species. Several systems of the infra-generic classification of Curcuma

Distribution and Species Diversity of Curcuma in Thailand 207

have been developed. Some of them are shown below.

Infra-generic classification of Curcuma

Baker (1892) Schumann (1904) Valeton (1918)
Subgenus Subgenus
Eucurcuma Eucurcuma
Section Section Section
Exantha Exantha Exantha
Section Section Section
Mesantha Mesantha Mesantha
Subgenus Subgenus
Section Hitcheniopsis Hitcheniopsis Paracurcuma

Baker (1890) divided Curcuma into three sections: Section Exantha
(the spikes separate from the shoot), Section Mesantha (the spikes borne
on the shoot either with or without leaves), and Section Hitcheniopsis
(characterized by autumnal spikes from the centre of the tuft of leaves;
bracts are very obtuse, adnate at the sides and spreading at the tip).

Schumann (1904) divided the genus into subgenus Eucurcuma and
raised the taxonomic rank of Hitcheniopsis Baker to subgenus. Subgenus
Eucurcuma is again divided into section Exantha and section Mesentha.

Valeton (1918) divided the genus into subgenus Eucurcuma and
Paracurcuma and divided the subgenus Eucurcuma into section Exantha and
Mesantha. Subgenus Paracurcuma was characterized by bracts connected at
least partly beyond the middle and often very numerous. Spike 1s cylindrical,
with comparatively short coma bracts. Anther spurs are very short (not a
quarter of the anther) or none.

However, more information for resolving the problems of
identification of this genus is still required.

Taxonomic Treatments

The previous taxonomic works on Curcuma from whole range of the
distribution of the genus are shown below. From these records and also
from recent collections from Thailand it can therefore be estimated , that
there are over 90 species of Curcuma in the world.

Baker (1890) recorded 29 species from India (10 species are not
found in Thailand).

Holttum (1950) reported 9 species from Malay Peninsula (1 species
is not found in Thailand.

208 Gard. Bull. Singapore 59 (1&2) 2007

Backer & Bakhuizen Van Den Brink (1963) reported 18 species
from Java (11 species are not found in Thailand).

Sabu and Mangaly (1996) presented 18 species from South India (8
species are added to the Baker’s list, and these are not found in Thailand).

Wu and Larsen (2000) published 12 species from China (6 species
are not found in Thailand).

Newman et al. (2004) reported 20 species from Malesia (16 species
are not found in Thailand).

Curcuma Species in Thailand

In Thailand, 38 species of Curcuma are now recognized. Among them,
Six species are undescribed (no. 33-38), three species are new records for
Thailand (11, 21, 25), three known species are endemic to Thailand (7,10
and 12) and eight species are cultivated for food and spices (1, 3,5, 9, 17, 18,
30, and 31).

1. C. aeruginosa Roxb. 21. C. pierreana Gagnep.

2. C.alismatifolia Gagnep. 22. C. rhabdota Sirirugsa & M.F. Newman
3. C.amada Roxb. 23. C. roscoeana Wall.

4. C. angustifolia Roxb. 24. C. rubescens Roxb.

5. C.aromatica Salisb. 25. C. rubrobracteata Skornick., M.
6. C.aurantiaca Zijp Sabu & Prasanthk.

7. C. bicolor Mood & K. Larsen 26. C. singularis Gagnep.

8. C.cochinchinensis Gagnep. 27. C. sparganiifolia Gagnep.

9. C.comosa Roxb. 28. C. stenochila Gagnep.

10. C. ecomata Craib 29. C. viridiflora Roxb.

11. C flaviflora S.Q. Tong 30. C. zanthorhiza Roxb.

12. C. glans K. Larsen & Mood 31. C. zedoaria (Christm.) Roscoe
13. C. gracillima Gagnep. 32. C. larsenii Maknoi & Jenjitt.
14. C. harmandii Gagnep. 33. C. sirirugsae (in prep.)

15. C. latifolia Roscoe 34. C.sp.

16. C. leucorhiza Roxb. 353 C2 Spi

17. C. longa L. SopC ap:

18. C. mangga Valeton & Zijp ST OMEASp:

19. C. parviflora Wall. 50. C. Sp:

20. C. petiolata Roxb.

Distribution and Species Diversity of Curcuma in Thailand 209

Infra-generic Classification of Curcuma in Thailand

Based on morphological characters, 38 species of Curcuma in Thailand
can be divided into 5 groups. Anther types of Curcuma are the important
distinctive characters for the classification into groups (Fig. 4).

A B Cc D BE

Figure 4. Anther types: A. “Alismatifolia” type; B. “Cochinchinensis” type; C. “Ecomata”
type; D. “Longa” type; E. “Petiolata” type.

Five Groups of Curcuma in Thailand

The distinguished characters, representative species, with short information
and illustrations of the five groups of Curcuma in Thailand are presented
below (Fig. 5).

1. “Alismatifolia” group

Distinguished characters are:

- Anther spurs absent;

- Stylodes absent.

Eight species are in this group: C. alismatifolia, C. gracillima, C. harmandii,
C. parviflora, C. rhabdota, C. sparganiifolia and two new species.

C. alismatifolia Gagnep.

This species is native to Thailand in the northeast and distributed to Laos
and Cambodia. It is commonly found in open areas in the pine or deciduous
forests at altitude 1300 m above sea level. C. alismatifolia has become an
important economic plant for the aesthetics of its inflorescences. It is easily
identified by the long, slender and stiff peduncle, the large and bright pink
coma bracts. The newly improved cultivars of this species, such as the white-
bract form, have also appeared in the markets for more than ten years
(Wanakrairote in 1996).

210 Gard. Bull. Singapore 59 (1&2) 2007

C. parviflora Wall.

This species was originally found in Myanmar and distributed throughout
tropical Asia. It grows in wide range of altitudes from 300 to over 1000 m
above sea level. It is also commercially popular for cut flowers or as potted
plants. The plant is small, about 30 cm tall. Its inflorescence is attractive with
the white coma bracts.

C. rhabdota Sirirugsa & M. F. Newman

This species was described in 2000 after it has become popular as ornamental
plant. It is widely spread both in Thailand and other countries. It was first
known from a selling at Chong Mek market at the Laos-Thai border in Ubon
Ratchathani province, and was collected from Laos as told by the seller.
This plant was brought to grow in the Royal Botanic Garden Edinburgh,
from which it was taken as the type specimen. However, it has been found
later that this species commonly grows in Ubon Ratchathani Province of
Thailand.

C. harmandii Gagnep.

This species is native to Cambodia and distributed to eastern, southeastern
and central Thailand. The uniqueness of its bracts with dark green, lanceolate
and reflexed apex is attractive and easily recognized for this species.

C. sparganiifolia Gagnep.

This is a native of Indochina and is distributed to northeastern, eastern and
southeastern Thailand. It is a small plant; its leafy shoot is about 15-20(-30)
cm tall. This species can be distinguished by its spike with slender peduncle,
pinkish-purple and suborbicular bracts.

2. “Cochinchinensis”’ group

Distinguished characters are:

- Anther spurs filamentous;

- Stylodes shortly cylindrical.

Two species are in this group: C. cochinchinensis and C. pierreana

C. pierreana Gagnep.

The species is native to Cambodia. In Thailand, it is found only in the
northeast. Its sessile inflorescence, white staminodes with large purple
blotches apices are distinctive characters for this species.

3. “Ecomata” group
Distinguished characters are:
- Anther spurs broad and blunt;
- Stylodes long and slender.

Distribution and Species Diversity of Curcuma in Thailand 214

Seven species are in this group: C. bicolor, C. ecomata, C. flaviflora, C. glans,
C. singularis, C. stenochila, and one new species)

C. bicolor Mood & K.Larsen

This species is endemic to Thailand, which was described in 2001. The plant
is 40-60 cm tall. It can be distinguished from other species in the “Ecomata”
group by its yellow staminodes with red blotches at the bases.

C. ecomata Craib
This is also endemic to Thailand. It is easily recognized by its purple labellum
with the dark yellow midband.

C. flaviflora S.Q. Tong

This is a Yunnan plant and distributed to northern Thailand. It grows at high
altitude from 1200-1400 m above sea level. This species can be distinguished
by its bright yellow flowers.

C. glans K.Larsen & Mood

This is another endemic species of Thailand that was recently described in
2001. Its yellow staminodes with red apices covering with densely glandular
hairs are the important distinctive characters of this species.

It is noted that the above four species of “Ecomata” group are
uncommon in Thailand.

4. “Longa” group
Distinguished characters are:

- Anther spurs acicular, inwardly curved;

- Stylodes cylindrical;

- Bract apex acute.
Thirteen species are in this group: C. aeruginosa, C. amada, C. angustifolia,
C. aromatica, C. comosa, C. latifolia, C. leucorrhiza, C. longa, C. mangga, C.
rubescens, C. viridiflora, C. zanthorrhiza, and C. zedoaria.

C. aeruginosa Roxb.

This is a native of Myanmar and distributed to India, Indochina, Malaysia,
Indonesia and Ceylon. In Thailand, it is commonly found in the dipterocarp
forests and is also cultivated. It is distinguished from other species by its
bluish-green rhizome and red corolla-lobes. The rhizome is medicinally used
throughout its range of distribution.

C. comosa Roxb.
The original country of this species is Myanmar, but is distributed to India,
and is cultivated in Malaysia. In Thailand, it is rarely found in the deciduous

212 Gard. Bull. Singapore 59 (1&2) 2007

and bamboo forests. It is commonly cultivated for medicinal purpose. This
species can be identified by its sessile inflorescence, white bracts tinted with
pink and white coma bracts with pink apex throughout dorsal midband.

C. longa L.

This species is well known as the “commercial turmeric”. Itis widely cultivated
in Asia. Turmeric is an important spice, which is used in the preparation of
curries in many Asian countries. It is also used in many other ways, such as
the source of yellow dye, cosmetic and medicines. Its coma bracts are mostly
white but vary to pale yellow or white with pink apices.

C. mangga Valeton & Zijp

This species is cultivated throughout Thailand. It is also commonly cultivated
in Malay Peninsula and Java. Coma bracts are pink or white, with a largely
pink blotch at the centre. This species is easily recognized by its white
rhizome that is pale yellow inside and the smell of mango. It is extensively
used as vegetable.

C. rubescens Roxb.

It is native to India, distributed to Myanmar, and uncommonly cultivated in
Thailand. Its red petioles and leaf-sheaths are good distinguishing characters
for this species.

C. zanthorrhiza Roxb.

This species was described by Roxburgh from a plant said to have been
introduced to Calcutta from Amboina, Moluccas (Holttum, 1950). It is
widely cultivated throughout SE Asia. Its rhizome is large, dark yellow
or yellowish-orange inside. It is used extensively in traditional medicine
particularly in Malaysia and Indonesia.

C. zedoaria (Christm.) Roscoe

A native species of India, it is cultivated throughout Southeast Asia. The
plant is about a meter tall, the primary rhizome is ovoid, and the inside of
the tuber is pale sulphur yellow. It is widely used as medicine in India and
Malaysia. The leaves are also used for flavouring fish and other foods in
Java.

5. “* Petiolata”’ group

Distinguished characters are:

- Anther spurs shortly acicular, straight;

- Stylodes cylindrical;

- Bract apex truncate or rounded.
Eight species are in this group: C. aurantiaca, C. petiolata, C. roscoeana, C.
rubrobracteata and four new species.

Distribution and Species Diversity of Curcuma in Thailand 213

C. aurantiaca Zijp

This is a native of Ceylon and is also present in Java and Malaysia. In
Thailand, it commonly grows in the clearing of evergreen forests and rubber
plantations in the south. This species can be recognized by its brownish-
green flower bracts, purplish-pink coma bracts, and anther without spurs. It
is also a well-known ornamental plant.

C. petiolata Roxb.

This species is distributed in India, Myanmar, Laos, Java and Thailand. It
can be identified by its large leaves with cordate base, pink coma bracts,
and white flower with yellow midband on the labellum. Its inflorescence
may be the largest of the genus. It is highly popular as an ornamental plant.
The variegated form of this species is called in Thai “Bua Chun”. Its flower
bracts are greenish with pink apices and its inflorescence is narrower. Note:

the hybrids between C. aurantiaca and C. petiolata have produced attractive
plants.

C. roscoeana Wall.

It is a native of Myanmar. In Thailand, it commonly grows in deciduous
forests and has been well known as an ornamental plant for a long time. This
species is easily identified by its orange flower bracts and anthers without
spurs.

C. rubrobracteata Skornick., M. Sabu & Prasanthk.

This species is distributed in India and Myanmar. In Thailand, it is commonly
found in deciduous and dry evergreen forests. The species was described in
2003, and is one of the new records for Thailand in 2005S.

Figure 5. A gallery of representative species of Curcuma in Thailand.

C. alismatifolia C. parviflora C. harmandii

Gard. Bull. Singapore 59 (1 &2) 2007

C . sparganiifolia C. rhabdota

C. glans
iif

C. aeruginosa C. comosa C. longa

Distribution and Species Diversity of Curcuma in Thailand

ae te,
r oa Pw
eee, be Z > ae

C. rubescens C. zanthorhiza

C. petiolata C. aurantiaca
(variegated form)

C. aurantiaca x petiolata C. roscoeana C. rubrobracteata

216 Gard. Bull. Singapore 59 (1&2) 2007

A Summary of Distribution of Curcuma in Thailand

‘“‘Alismatifolia” group has its centre of distribution in the northeastern and
eastern regions of the country.

‘“‘Cochinchinensis” group consists of only 2 species: one (C. cochinchinensis)
occurs in the north and southwest, another one (C. pierreana) has its limited
distribution in the eastern region.

“Ecomata” group has all its species occurring in the northern region;
however, two of them, C. singularis and C. stenochila, are also distributed in
the northeastern, eastern and southeastern parts of the country.

“Longa” group has most of its member species cultivated. In Thailand, few
of them grow wild in the north.

‘“*Petiolata’” group has most species distributed from the north to the
peninsula along the western ranges (Fig. 6).

Ginn,

siseeeestadas **A lismatifolia”

=—_=— = = “Cochinchinensis”

= * = Scomse
“Longa”

—s = “eno

4
v

. nt "4
. alae AL?

Figure 6. Distribution of Curcuma in Thailand

Distribution and Species Diversity of Curcuma in Thailand 217

Phylogenetic Relationship of Curcuma Species in Thailand

The strict concensus of three most parsimonious cladograms, which resulted
from ITS sequence analysis (Fig.7) reveals that three groups (“Alismatifolia”,
“Cochinchinensis” and “Ecomata’”’) are clearly separated from each other
and from the “Longa” group. The relationship of “Petiolata” group 1s
unclear. The study shows that except for “Petiolata” group, the remaining
four groups proposed by the morphological classification are supported by
molecular evidence. It also suggests that the new classification of the genus
Curcuma should be considered.

ml “ Alismatifolia’ |

100

N| ‘Longa’

Cur peti ry) ‘ :
™~ |||. ‘Petiolata’
98 74 Par bhati s ‘
r a

\\ ‘Ecomata’
AN

[ie

(
OxaT SRI Ni BSia0e Vor ow. Bick: i), ‘Cochinchinensis’

Hed villo

Figure 7. Cladogram showing the relationships of some Curcuma species (Maknoi, 2005)

Conclusion
Some suggestions for further studies on the genus Curcuma are:

1. Though several classification systems of Curcuma, as well as one from
this paper, have been proposed, it seems that the taxonomic problems of
this genus still exist. Further studies for more information, particularly the
phylogenetic relationships of species, are needed and required to support
the taxonomy of the grouping of this genus.

218 Gard. Bull. Singapore 59 (1&2) 2007

2. Regarding the diversity of the genus, it is believed that more undescribed
species of Curcuma can still be found in natural forests of tropical Asia.
Therefore, more explorations for new taxa are suggested.

3. Curcuma is a genus very useful to man. Many species are used as
medicines, ornamental plants, dyes, cosmetics, foods and spices. It is reported
that less than 50 % of the species are used by man. The rest, more than 50
% of Curcuma species have not been known of their uses. Therefore, more
studies on the biological activities and pharmacological actions of Curcuma
are needed in order to explore and search for their potential uses. Species
with high potential use in decoration are also suggested to be commercially
developed.

However, on account of conservation, over exploitation or over
collection of plants from the wild may cause the genetic erosion. The
awareness and warning of over using some species should be heeded.

References

Backer, C.A. and Bakhuizen Van Den Brink, R.C. 1963. Monocotyledones,
pp. 1-641, In: Flora of Java, vol. 3. N.V.P. Noordhoff-Groningen, The
Netherlands.

Baker, J.G. 1890. Scitamineae, pp. 198-264. In: Hooker, J.D., Flora of British
India, vol. 6. London.

Burtt, B.L. and Smith, R.M. 1972. Key Species in the taxonomic history of
Zingiberaceae. Notes from the Royal Botanic Gardens Edinburgh 31:
477-227.

Gagnepain, F. 1908. Zingiberacées, pp. 25-121. In: Gagnepain, F(ed.), Flore
Génerale de L’ Indo-Chine. Vol. 6. Masson et Cie, Paris.

Holttum, R.E. 1950. The Zingiberaceae of the Malay Peninsula. Gardens
Bulletin Singapore 13: 1-249.

Kress, W.J., L.M. Price and K.J. Williams. 2002. The phylogeny and a new
classification of the gingers (Zingiberaceae): Evidence from molecular
data. American Journal of Botany 89: 1682-1696.

Larsen, K. 2005. Distribution patterns and diversity centres of Zingiberaceae
in SE Asia. Biologiske Skrifter 55: 219-228.

Distribution and Species Diversity of Curcuma in Thailand 219

Maknoi, C. 2005. Taxonomy and Phylogeny of the genus Curcuma L.
(Zingiberaceae) with particular reference to its occurrence in Thailand.
Ph.D. Thesis. Prince of Songkla University.

Mood, J.D. and Larsen, K. 2001. New Curcuma from Southeast Asia. The
New Plantsman 8: 207-217.

Newman, M., A. Lhuillier and A.D. Poulsen. 2004. Checklist of the
Zingiberaceae of Malesia. Blumea 16: 65-69.

Sabu, M. & Mangaly, J.K. 1996. Taxonomic Revision of South Indian
Zingiberaceae, pp. 15-22. In: Wu,D-L., Wu, Q.-G, & Chen, Z.-Y (eds.).
Proceedings of the 2° Symposium on the Family Zingiberaceae.
Zhongshan University Press, Guangzhou.

Schumann, K. 1904. Zingiberaceae. In: Engler, A.(ed.), Das Pflanzenreich.,
IV, 46. Engelmann, Leipzig. 458pp.

Sirirugsa, P. and Newman, M. 2000. A new species of Curcuma L.
(Zingiberaceae) from SE. Asia. The New Plantsman 4: 196-198.

Valeton, T. 1918. New notes on the Zingiberaceae of Java and Malaya.
Bulletin du Jardin Botanique Buitenzorg ser. 2,27: 1-166.

Wanakrairote, S. 1996. Curcuma. Amarin Printing And Publishing Public
Co., Ltd. Bangkok.

Wu, D.-L. and Larsen, K. 2000. Zingiberaceae, pp.322-377. In: Wu, Z-Y.
& Raven, P.H.(eds.), Flora of China, vol. 24. Science Press, Beijing &
Missouri Bot. Gard. Press, St. Louis.

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Gardens’ Bulletin Singapore 59 (1&2): 221-230. 2007 oO4

Floral Ontogeny in Alpinia oxyphylla Miq.
(Zingiberaceae) and Its Systematic Significance

J.-J.SONG ‘2, P ZOU |, J-P. LIAO '*, Y.-J. TANG '!AND Z.-Y. CHEN |

‘South China Botanical Garden, Chinese Academy of Sciences,
Guangzhou 510650, China
* College of life science, Shenzhen University, Shenzhen 518060, China
* Corresponding author: Liaojp@scbg.ac.an

Abstract

Floral organ development of Alpinia oxyphylla Migq. begins with the initiation
of sepal primordia, and then the three common primordia comprising petal
and inner whorl androecial members. Each common primordium separates
into a dorsal petal and a ventral androecial member. The adaxial common
primordium begins to separate first to produce the functional stamen and
the adaxial petal. Subsequently the two abaxial common primordia separate
to form two abaxial inner androecial members and two abaxial petals. After
the three common primordia completed their differentiation, three outer
androecial members are formed, of which the two adaxial primordia have
a Slow growth and finally become two lateral staminodes, while the abaxial
primordium ceases growth and disappears gradually. The gynoecium is
the last floral structure to initiate. Soon after the initiation of gynoecial
primordium, the two abaxial inner androecial members form two secondary
primordia. Compared to the development of the anther on the fertile
stamen, the secondary primordia may be homologous with the primordia
of the pollen sacs. This provides new evidence supporting the view that the
labellum was derived from the two inner whorled androecial members.

Introduction

The Zingiberales is a natural order of monocotyledons consisting of eight
families, namely, Heliconiaceae, Strelitziaceae, Lowiaceae, Musaceae, Zin-
giberaceae, Costaceae, Cannaceae and Marantaceae (Tomlinson, 1982;
Dahlgren and Rasmussen, 1983). Among the eight families, Heliconiaceae,
Strelitziaceae, Lowiaceae and Musaceae are often informally referred to as
the banana group, and flowers of those four families possess either five or six
fertile stamens. The remaining four families form the monophyletic ginger
group. Flowers in the ginger families possess either one fertile stamen with

227, Gard. Bull. Singapore 59 (1&2) 2007

two anther sacs (Zingiberaceae and Costaceae) or one stamen with only
one anther sac (Marantaceae and Cannaceae) (Kress et al., 2001; Rudall
and Bateman, 2004). A typical flower of angiosperm consists of four whorls
of different floral organs, they are from outside to inside: sepals in whorl 1,
petals in whorl 2; stamens in whorl 3 and carpels in whorl 4. Nevertheless,
there are labellum and lateral staminodes in the whorl 3 besides the sta-
men in ginger group. Fig. 1 shows a flower diagram of Zingiberaceae and a
picture of mature flower of Alpinia oxyphylla Mia. to indicate the positions
of lateral staminodes and labellum. Inspite of several opinions about the
nature of the labellum and the lateral staminodes in the ginger family based
primarily on morphological and anatomical evidences expressed in the past
(e.g. Burrt, 1972; Gregory, 1936; Rao, 1963; Raghavan and Venkatasubban,
1941; Willis, 1948), the recent evidences support labellum being derived by
the congenial fusion of two inner staminodes (e.g. Kirchoff, 1997, 1998).

In angiosperms, many structures that are absent in mature flower
have vestigial remains that can be observed during the development of
floral primordium (Endress, 1994). Accordingly, floral organogenesis and
development can reveal not only the early state of the floral structure in
the flower, but also the homeosis of floral structure. Kirchoff has described
the aspect of floral development of several species in the Zingiberaceae
(Kirchoff, 1988a, 1997, 1998) and other Zingiberales (Kirchoff, 1983, 1986,
1988b), and Box and Rudall (2006) have investigated the floral ontogeny in
Globba.

Reseseerssseesrers,
a

Lateral
Staminode

Latera! Staminode Lateral Staminode
Labellum

Staminode § > Absent Stamen

Figure 1. Floral diagram of Zingiberaceae and a mature flower of Alpinia oxyphylla. A.
Floral diagram of Zingiberaceae (cited from Kress et al, 2002 ); B. mature flower of Alpinia
oxyphylla.

Floral Ontogeny in Alpinia oxyphylla 223

In the genus Alpinia only one species, Alpinia calcarata (Haw.)
Roscoe, was studied (Kirchoff, 1988a). Here we describe the floral
development of Alpinia oxyphylla to provide additional information for the
species within the genus A/pinia and present additional evidence to a better
understanding of the origin of the labellum and the lateral staminodes in the
family Zingiberaceae.

Materials and Methods

Inflorescences of Alpinia oxyphylla were collected from the ginger garden
of South China Botanical Garden, Chinese Academy of Sciences. Fresh
materials were fixed using forlmalin-acetic acid-alcohol (FAA) (Berlyn and
Miksche, 1976) for two days, and then stored in 70% alcohol. The vouchers
(collection number: J. J. Song 2004-03) were deposited at SCBG. Specimens
used for scanning electron microscope (SEM) were dissected in 70% alcohol
under Wild Stereo Microscope, dehydrated in a series of alcohol running up
to 100%, treated with isoamyl acetate, and then critical-point dried using
CO, Gold-sputtered specimens were observed and photographed under
the SEM.

Results

The shoot apex of Alpinia oxyphylla is a domed shape and more or less
symmetrical structure and produces leaves in an alternate arrangement (Fig.
2). A crescent-shaped primordium initiates on one side of the apex, which
indicates the initiation of a leaf. This leaf primordium has a rapid growth
and forms a cap-like structure; gradually encloses the apex from above.
When this cap-like structure encloses half of the shoot apex, another foliar
primordium begins to initiate on the other side of the apex and repeats the
same growth process (Fig. 2). After about the differentiation of 17-20 leaves,
the shoot apical meristem converts itself into an inflorescence meristem.

The inflorescence of A. oxyphylla is a raceme (Wu and Larsen,
2000) and there are about 50 to 70 flowers that are initiated in acropetal
order in one inflorescence (Fig. 3). Floral organ development begins with
the initiation of sepal primordium. The three sepal primordia are initiated
sequentially at the three angles of the floral shoot apex (Figs. 4, 5). These
three primordia continue to enlarge and gradually separate from the central
part of the floral primordia to form a ring of calyx primordium (Figs. 6, 7,
8).

Soon a bulge initiates at the adaxial side of the midsection of the
floral primordium after the initiation of the sepal primordium (Fig. 5).

224 Gard. Bull. Singapore 59 (1&2) 2007

100nm

as ? ~ SMR wm - -

Figures 2-9. Floral organogeny of Alpinia oxyphylla: 2. The vegetative apex (va) and the foliar
primordium (fp). The black arrow indicates the initiation of a new foliar primordium. 3. The
inflorescence shoot. 4. The first sepal (c1) is initiated on a flower bud. 5. Sepals (cl, c2) labeled
in order of initiation; cpl, cp2: the two primordia of petal and inner whorl androecium. 6.
Three sepals (cl,c2, c3) begin to form a ring calyx primordium. 7, The complete formation
of three primordia (cp) labeled in order of initiation. 8, The three primordia separate into
petal and inner whorled androecium (ia) [p, petal; st, stamen; ia, abaxial inner whorled
androecial member]. Black arrows indicate pollen sac of the anther. 9. Formation of three
outer androecial members (0a) [p, petal; st, stamen; ia, abaxial inner whorled androecial
member]. Black arrows indicate pollen sacs.

Thereafter, two other bulges form in a counter-clockwise order at the abaxial
side of the floral primordium (Figs. 5, 6). These are the common primordia
of three petals and the three inner whorl androecial members. The three
common primordia arrange triangularly and fuse basally to form the floral
cup. However, there is an unequal development of these three primordial:
the adaxial one becomes obviously larger than the abaxial two (Fig. 7). The
center of the floral cup is depressed, and the depression becomes deeper
with the growth of the three common primordia (Figs. 6, 7). The three petals
and their associated androecial members are formed from the separation

Floral Ontogeny in Alpinia oxyphylla 223

of these three common primordia. Each primordium separates into a
dorsal petal and a ventral inner whorled androecial member. The adaxial
primordiun begins to separate first and produces the functional stamen and
the adaxial petal. Subsequently the two abaxial primordia separate more
or less simultaneously to form the two abaxial inner androecium and two
abaxial petals (Fig. 8).

Additionally, the primordium of the fertile stamen differentiates
into two bulges soon after its formation and these two bulges grow rapidly
to give rise to the locules (pollen sac) of the anther (Figs. 8, 9, see arrows).
The two abaxial inner androecial primordia then fuse with each other, and
ultimately form the labellum. After the three common primordia complete
their differentiation, three outer androecial members begin to form (Fig.
9): two of them appear beside the stamen primordium, one presents at
the position outside the fused part of the two abaxial inner androecial
primordia. The two adaxial primordia of the outer androecial members
form the lateral staminodes when the flower finishes differentiation (Fig. 15,
see black arrow). The abaxial one ceases growth soon after its initiation and
disappears gradually (Figs.10-12, see black arrows).

The three petals undergo rapid growth after their initiation and
enclose the inner floral organs gradually. The floral primordium has
differentiated calyx, petal, inner and outer whorl androecial members at this
stage. The gynoecial primordium is the last one to initiate; it appears in the
depression formed by the development of outer whorls (Fig. 10). The gynoecial
primordium grows rapidly following initiation and soon forms the stigma
and style (Figs. 13-15). Soon after the initiation of gynoecial primordium, the
two abaxial inner androecial primordia, which ultimately form the labellum,
each begin to produce two secondary primordia respectively (Fig.11, see
white arrows). The size of the four secondary primordia is similar when
they are just formed (Fig.11), but there is an unequal development among
them. The abaxial two grow faster than the adaxial two, which results in the
difference of their sizes (Fig.12). Furthermore, the difference between their
sizes becomes greater with growth and it is these four secondary primordia
eventually form the semi-oval labellum when all floral organs finish the
ontogenetic differentiation (Figs. 12-15).

Discussion

Kirchoff (1983, 1986, 1988a, b, 1997, 1998) studied the floral organogenesis
in Zingiberales and established the pattern of floral ontogeny in the order,
which in general, is highly conserved at the family level. The floral organ
development of A. oxyphylla fits well into this general pattern.

226 Gard. Bull. Singapore 59 (1&2) 2007

Figures 10-15. Floral organogeny of Alpinia oxyphylla: 10. The initiation of the gynoecium
(g). Black Arrow indicates the abaxial outer whorled androecial member. 11. Two abaxial
inner androecial members begin to form secondary primordia (white arrows) [oa, outer
whorl androecium]. 12-13. Size differentiation (see white arrows) between the secondary
primordia of inner androecium. 14. The differentiation of stigma (sti) and style (sty). 15, The
adaxial outer whorl androecium with subulate appendage (see arrow).

The initiation sequence of the floral organ is sepal, petal and inner
androecium, outer androecium, gyncecial primordium, which resembles the
developmental pattern reported for A. calcarata (Kirchoff, 1988a). In our
study of A. oxyphylla, some of the developmental characters observed are
like A. calcarata: (1) there is a lag between the formation of the inner and
outer androecial whorls, that is, the outer androecial members do not begin to
initiate until after the petals and the inner androecium are distinctly formed;
(2) the three common primordia initially are asymmetric, and originated on
the adaxial side of the floral shoot apex; and (3) the shape of the floral cup.
However, these characters observed in the floral ontogeny of two species of
Alpinia are different or partly different from what was described by Kirchoff
(1988a) for other members of the ginger group. Kirchoff (1988a) had used
the characters of floral ontogeny in his phylogenetic analysis of the family.
Our data on the floral ontogeny may add information to the understanding
of the relationship between the floral development and evolution of the
individual groups in the family.

The formation of the labellum and the lateral staminodes of species
in Zingiberaceae has received much attention. Various interpretations have
been advanced. Brown (1830) regarded the labellum and the two subulate

Floral Ontogeny in Alpinia oxyphylla aa |

appendages as the outer whorled stamens, and the two epigynous glands
and the functional stamen as the inner whorled stamens. Raghavan and
Venkatasubban (1941) had the same point of view on the basis of work
on A. calcarata. But Rao (1963) proved that the two epigynous glands of
Zingiberaceae are merely an outgrowth from the upper surface of the
ovary. Gregory (1936) gave an interpretation that the stamen and the lateral
portions of the labellum belong to the inner whorl, while the median part of
the labellum and the two subulate appendages belong to the outer whorl on
the basis of his work on Elettaria cardamomum (L.) Maton. Others, like Willis
(1948), believed that the functional stamen and the labellum represented the
inner whorled stamens. Liao et al. (2006) studied the floral vasculature of
Alpinia hainanensis and showed that the labellum is supposed to represent
five members of the androecium: its two marginal and the median portions
are derived from three members of the outer androecial whorl and its two
lateral parts represent the two members of the inner whorl. The recent
evidences supporting the origin of labellum is derived from the congenial
fusion of two staminodes, and the two lateral staminodes represent the outer
androecial whorl, the anterior member of this whorl being absent.

The floral development study reported by Kirchoff (1997, 1998)
supported the interpretation that the primordia of the two inner staminodes
are joined by the intercalary growth to produce the labellum, while the
abaxial outer androecial member ceases growth soon after initiation and
contributes only initially to the formation of the labellum. The other two
outer androecial members form the two lateral staminodes. In our study,
the floral development of A. oxyphylla also supports this interpretation.
Moreover, the two abaxial inner androecial primordial differentiated into
two secondary primordia respectively. Compared with the development
of the anther on the fertile stamen, the two secondary primordia maybe
homologous to the primordia of two locules (pollen sac) of the anther.
From this point of view, the four secondary primordia observed by us in
A. oxyphylla that eventually form the labellum, may represent the fusion
of four pollen sacs of two stamens. This brings forth new evidence for the
view that the labellum was derived from the two inner whorled androecial
members.

Acknowledgements

This research was supported by National Natural Science Foundation of
China (39870087, 30370099, 40332021) and National Key Program for Basic
Research of China (2001CCA00300).

228 Gard. Bull. Singapore 59 (1&2) 2007

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